Treatment of leg ulcers using placenta derived collagen biofabric

ABSTRACT

The present invention provides a method of treating a leg ulcer comprising contacting the leg ulcer with a collagen biofabric. The leg ulcer may be a venous, arterial, diabetic or decubitus (pressure) ulcer. The invention further provides kits comprising one or more pieces of collagen biofabric for the treatment of a leg ulcer.

This application claims benefit of U.S. Provisional Application Ser. No.60/699,441, filed Jul. 13, 2005, which is hereby incorporated byreference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to methods and compositions for thetreatment and repair of leg ulcers, particularly venous leg ulcers,using a placenta-derived collagen biofabric.

2. BACKGROUND OF THE INVENTION

Leg ulcers, which are breaks in the skin of the leg, especially aroundthe ankles, and foot ulcers, are a persistent problem in certain patientpopulations, particularly affecting elderly, immobile, obese,under-exercised, diabetic or atherosclerotic patients. Chronic legulcers are persistent, non-healing or slow-healing wounds in which thebody's healing process becomes stalled. The four most common types ofleg ulcers are: venous leg ulcers, arterial leg ulcers, diabetic ulcers,and decubitus (pressure) ulcers.

Leg ulcers, particularly venous leg ulcers (also known as venous stasisulcers), have been treated by application of a variety of skinsubstitutes or dressings. An example of a collagen dressing is OASIS®,an extracellular matrix product derived from porcine small intestinesubmucosa. APLIGRAF®, a living bi-layered skin substitute, comprises adermal layer and an epidermal layer. ALLODERM®, a dermal matrix product,is a tissue derived from donated human skin. The skin substitutes,however, have disadvantages, including high expense, difficulty inhandling, delicacy or difficulty in obtaining graft material.

Thus, despite the prevalence of leg ulcerations, particularly venous legulcerations, and the availability of types of graft materials, thereexists a need for a method of healing leg ulcers using a relativelyinexpensive, lightweight, readily-available, durable material that canfacilitate healing of the ulcerated area.

3. SUMMARY OF THE INVENTION

The present invention provides methods of treating a leg ulcer,comprising contacting said ulcer with a collagen biofabric for a timesufficient to improve at least one aspect of the leg ulcer, or preventor reduce the worsening of at least one aspect of a leg ulcer. In oneembodiment, said contacting is for a time sufficient for at least oneaspect of the leg ulcer to measurably improve compared to a leg ulcernot contacted with the collagen biofabric. In another specificembodiment, said contacting is for a time sufficient to prevent orreduce the worsening of at least one aspect of a leg ulcer, compared toa leg ulcer not contacted with the collagen biofabric. In anotherspecific embodiment, said contacting comprises placing the collagenbiofabric on the leg ulcer so that substantially all of the surface areaof the biofabric contacts the leg ulcer. In another specific embodiment,said contacting comprises placing the collagen biofabric on a meshedskin graft covering a leg ulcer so that a plurality, or substantiallyall, of the interstices of the skin graft are in contact with thebiofabric. In various embodiments, said leg ulcer can be, for example, avenous leg ulcer, arterial leg ulcer, diabetic leg ulcer, decubitusulcer, or split thickness skin grafted ulcer or wound.

The preferred collagen biofabric is substantially dry, i.e., about 20%or less water by weight, and substantially retains the proteins of theamnion, chorion, or both in their native, unmodified conformations. In aspecific embodiment, for example, the collagen biofabric is notprotease-treated. In another specific embodiment, proteins within saidcollagen biofabric are not artificially chemically crosslinked, that is,the collagen biofabric is not fixed. In another specific embodiment, thecollagen biofabric is substantially dry prior to said contacting. Inanother specific embodiment, said collagen biofabric is provided as asheet, membrane, netting or webbing. In another specific embodiment, thecollagen biofabric is diced or sheared such that it is flowable througha cannula or needle. In another specific embodiment, said collagenbiofabric is mounted on a support. In a more specific embodiment, saidsupport is a bandage.

In another specific embodiment, said collagen biofabric additionallycomprises a bioactive compound not naturally-occurring in the collagenbiofabric, or present in a different concentration than in collagenbiofabric to which the bioactive compound has not been added. In a morespecific embodiment, said bioactive compound is a small organicmolecule, a mineral, a metal, an antibiotic, amino acid, painmedication, anti-inflammatory agent, cytokine, growth factor, enzymeinhibitor, kinase inhibitor, an anti-tumor agent, an anti-fungal agent,an anti-viral agent, an anti-infective agent, a wound sealant or a woundhealing or sealing agent. In a more specific embodiment, said woundhealing or sealing agent is platelet-derived growth factor (PDGF),transforming growth factor (TGF), lactoferrin, thymosin, hyaluronic acid(HA), fibrin, fibronectin or thrombin, or a combination thereof.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Treatment of Leg UlcersUsing Collagen Biofabric

The present invention provides methods for the treatment of a leg ulcerusing a collagen biofabric. In this context, “treatment of a leg ulcer”comprises contacting the leg ulcer with collagen biofabric for a timesufficient to improve at least one aspect of the leg ulcer, or to reducethe worsening of at least one aspect of the leg ulcer, as compared to aleg ulcer not contacted with collagen biofabric. As used herein, “aspectof the leg ulcer” includes objectively measurable parameters such asulcer size, depth or area, degree of inflammation, ingrowth ofepithelial and/or mesodermal tissue, gene expression within theulcerated tissue that is correlated with the healing process, qualityand extent of scarring etc., and subjectively measurable parameters,such as patient well-being, perception of improvement, perception oflessening of pain or discomfort associated with the ulcer, patientperception that treatment is successful, and the like.

Thus, in a preferred embodiment, the invention provides a method oftreating a leg ulcer comprising contacting the leg ulcer with collagenbiofabric for a sufficient time to heal the ulcer, e.g., for a timesufficient for the epithelium to close over the ulcer. In anotherpreferred embodiment, the leg ulcer is contacted with collagen biofabricfor a time sufficient for at least one aspect of the leg ulcer to showmeasurable improvement, compared to a leg ulcer not contacted withcollagen biofabric. In another embodiment, the leg ulcer is contactedwith collagen biofabric for a time sufficient to prevent, or reduce, theworsening of at least one aspect of the leg ulcer, compared to a legulcer not contacted with the collagen biofabric.

Generally, the collagen biofabric is contacted with the leg ulcer in anymanner that maximizes contact between the leg ulcer and collagenbiofabric. For example, the collagen biofabric can be laid or pressedgently as a dry sheet on the leg ulcer, and can be allowed to adhere tothe leg ulcer by contact alone. The dry sheet may optionally then bewetted so that the collagen biofabric additionally adheres to the skinsurrounding the ulcer. In another embodiment, the collagen biofabric canbe pre-wetted, for example with a saline solution, and laid or pressedgently onto the leg ulcer. As in the previous embodiment, the biofabriccan be allowed to adhere to the leg ulcer without external means ofsecuring the biofabric. In another embodiment, the leg ulcer andcollagen biofabric can be covered by a bandage such that the collagenbiofabric is pressed onto the surface of the leg ulcer. In a specificembodiment, the collagen biofabric can be attached to, or adhered to,the bandage. The collagen biofabric may, less preferably, be sutured,glued or stapled to tissue surrounding the leg ulcer. During the courseof treating a leg ulcer, collagen biofabric may be applied a pluralityof times. For example, when collagen biofabric contacted with a legulcer becomes absorbed into the ulcer, or otherwise degraded, the legulcer can be contacted with a fresh piece of the collagen biofabric.Preferably, the collagen biofabric covers all or substantially all ofthe surface of the leg ulcer.

In preparation for, and during, contacting a leg ulcer with collagenbiofabric, the ulcer is preferably kept moist. Dry ulcers can be wettedwith, e.g., a saline solution prior to contacting with the collagenbiofabric.

The invention provides for the treatment of a leg ulcer using collagenbiofabric at any point in the development and pathogenesis and/orhealing of the ulcer.

Venous Leg Ulcers

The invention provides for the treatment of venous leg ulcers usingcollagen biofabric. Venous leg ulcers, also known as venous stasisulcers or venous insufficiency ulcers, a type of chronic or non-healingwound, are widely prevalent in the United States, with approximately 7million people, usually the elderly, afflicted. Worldwide, it isestimated that 1-1.3% of individuals suffer from venous leg ulcers.Approximately 70% of all leg ulcers are venous ulcers. Venous leg ulcersare often located in the distal third of the leg known as the gaiterregion, and typically on the inside of the leg. The ulcer is usuallypainless unless infected. Venous leg ulcers typically occur because thevalves connecting the superficial and deep veins fail to functionproperly. Failure of these valves causes blood to flow from the deepveins back out to the superficial veins. This inappropriate flow,together with the effects of gravity, causes swelling and progression todamage of lower leg tissues.

Patients with venous leg ulcers often have a history of deep veinthrombosis, leg injury, obesity, phlebitis, prior vein surgery, andlifestyles that require prolonged standing. Other factors may contributeto the chronicity of venous leg ulcers, including poor circulation,often caused by arteriosclerosis; disorders of clotting and circulationthat may or may not be related to atherosclerosis; diabetes; renal(kidney) failure; hypertension (treated or untreated); lymphedema(buildup of fluid that causes swelling in the legs or feet);inflammatory diseases such as vasculitis, lupus, scleroderma or otherrheumatological conditions; medical conditions such as high cholesterol,heart disease, high blood pressure, sickle cell anemia, or boweldisorders; a history of smoking (either current or past); pressurecaused by lying in one position for too long; genetics (predispositionfor venous disease); malignancy (tumor or cancerous mass); infections;and certain medications.

Venous ulcers are typically treated with wound debridement and withcompression of the leg to minimize edema or swelling. Compressiontreatments include wearing therapeutic compression stockings, multilayercompression wraps, or wrapping an ACE bandage or dressing from the toesor foot to the area below the knee. An Unna boot may be used, but isless preferred.

Thus, in another embodiment, the invention provides a method of treatinga venous leg ulcer comprising contacting the venous leg ulcer withcollagen biofabric and applying compression to the venous leg ulcer, fora time sufficient to improve at least one aspect of the venous legulcer, or to lessen the worsening of at least one aspect of a venous legulcer, as compared to a venous leg ulcer not contacted with collagenbiofabric. In a specific embodiment, said compression is applied by atherapeutic compression stocking, multilayer compression wrap, ACEbandage, or Unna boot. In a specific embodiment, the ulcer is debridedprior to contacting with the collagen biofabric. In another specificembodiment, the method additionally comprises treating an underlyingcause of the venous leg ulcer.

Other Leg Ulcer Types

Arterial leg ulcers are caused by an insufficiency in one or morearteries' ability to deliver blood to the lower leg, most often due toatherosclerosis. Arterial ulcers are usually found on the feet,particularly the heels or toes, and the borders of the ulcer appear asthough they have been ‘punched out’. Arterial ulcers are frequentlypainful. This pain is relieved when the legs are lowered with feet onthe floor as gravity causes more blood to flow into the legs. Arterialulcers are usually associated with cold white or bluish, shiny feet.

The treatment of arterial leg ulcers contrasts to the treatment ofvenous leg ulcers in that compression is contraindicated, as compressiontends to exacerbate an already-poor blood supply, and debridement islimited, if indicated at all. Thus, in another embodiment, the inventionprovides a method of treating an arterial leg ulcer comprising treatingthe underlying cause of the arterial leg ulcer, e.g., arteriosclerosis,and contacting the arterial leg ulcer with collagen biofabric for a timesufficient to improve at least one aspect of the arterial leg ulcer, orto lessen the worsening of at least one aspect of the arterial legulcer, as compared to an arterial leg ulcer not contacted with collagenbiofabric. In a specific embodiment, the method of treating does notcomprise compression therapy.

Diabetic foot ulcers are ulcers that occur as a result of complicationsfrom diabetes. Diabetic ulcers are typically caused by the combinationof small arterial blockage and nerve damage, and are most common on thefoot, though they may occur in other areas affected by neuropathy andpressure. Diabetic ulcers have characteristics similar to arterialulcers but tend to be located over pressure points such as heels, ballsof the feet, tips of toes, between toes or anywhere bony prominences rubagainst bed sheets, socks or shoes.

Treatment of diabetic leg ulcers is generally similar to the treatmentof venous leg ulcers, though generally without compression;additionally, the underlying diabetes is treated or managed. Thus, inanother embodiment, the invention provides a method of treating adiabetic leg ulcer comprising treating the underlying diabetes, andcontacting the diabetic leg ulcer with collagen biofabric for a timesufficient to improve at least one aspect of the diabetic leg ulcer, orto lessen the worsening of at least one aspect of the diabetic legulcer, as compared to a diabetic leg ulcer not contacted with collagenbiofabric.

Decubitus ulcers, commonly called bedsores or pressure ulcers, can rangefrom a very mild pink coloration of the skin, which disappears in a fewhours after pressure is relieved on the area to a very deep woundextending into the bone. Factors known to be associated with thedevelopment of ulcers include advanced age, immobility, poor nutrition,and incontinence. Stage 1 decubitus ulcers exhibit nonblanchableerythema of intact skin. Stage 2 decubitus ulcers exhibit superficial orpartial thickness skin loss. Stage 3 decubitus ulcers exhibit fullthickness skin loss with subcutaneous damage. The ulcer extends down tounderlying fascia, and presents as a deep crater. Finally, stage 4decubitus ulcers exhibit full thickness skin loss with extensivedestruction, tissue necrosis, and damage to the underlying muscle, bone,tendon or joint capsule.

After removing the pressure or abrasion that is the underlying cause ofthe decubitus ulcer, treatment generally involves keeping the areaclean, promoting tissue regeneration, and removing dead tissue. Thus, inanother embodiment, the invention provides a method of treating adecubitus leg ulcer comprising removing the pressure causing thedecubitus ulcer, and contacting the decubitus leg ulcer with collagenbiofabric for a time sufficient to improve at least one aspect of thedecubitus leg ulcer, or to lessen the worsening of at least one aspectof the decubitus leg ulcer, as compared to a decubitus leg ulcer notcontacted with collagen biofabric.

A preferred collagen biofabric for use in the methods of treatment of aleg ulcer, described herein, is a collagen-containing, placenta-derivedamniotic and/or chorion membrane material used as a film, membrane, orsheet. A particularly preferred collagen biofabric is the vacuum-dried,non-fixed, non-protease-treated amniotic membrane material described inHariri, U.S. Application Publication U.S. 2004/0048796, which is herebyincorporated in its entirety, and produced by the methods describedtherein, and herein (see Examples 1, 2).

The invention further encompasses treating a leg ulcer using collagenbiofabric in conjunction with one or more therapies or treatments usedin the course of treating a leg ulcer. The one or more additionaltherapies may be used prior to, concurrent with, or after use of thecollagen biofabric. The collagen biofabric, and one or more additionaltherapies, may be used where the collagen biofabric alone, or the one ormore additional therapies, alone, would be insufficient to measurablyimprove, maintain, or lessen the worsening of, one or more aspects of aleg ulcer. For example, the invention provides for the treatment of aleg ulcer comprising contacting the leg ulcer with a collagen biofabric,and treating the leg ulcer in another manner not comprising contactingthe leg ulcer with a collagen biofabric, where the contacting and thetreating the leg ulcer using an additional therapy together cause ameasurable improvement in, maintenance of, or lessening of the worseningof, at least one aspect of a leg ulcer, as compared to a leg ulcer notcontacted with a collagen biofabric. In specific embodiments, the one ormore additional therapies comprise, without limitation, treatment of theleg ulcer with a wound healing agent (e.g., PDGF, REGRANEX®);administration of an anti-inflammatory compound; administration of apain medication; administration of an antibiotic; administration of ananti-platelet or anti-clotting medication; application of a prosthetic;application of a non-collagen biofabric dressing (e.g., moist to moistdressings; hydrogels/hydrocolloids; alginate dressings; collagen-basedwound dressings; antimicrobial dressings; composite dressings; syntheticskin substitutes, etc.), and the like. In another embodiment, theadditional therapy comprises contacting the leg ulcer with honey. Forany of the above embodiments, in a specific embodiment, the leg ulcer isa venous leg ulcer, a decubitus ulcer, a diabetic ulcer, or an arterialleg ulcer.

In another specific embodiment, the additional therapy is a painmedication. The invention thus provides a method of treating a leg ulcercomprising contacting the leg ulcer with a collagen biofabric, andadministering a pain medication to lessen or eliminate leg ulcer pain.In a specific embodiment, the pain medication is a topical painmedication.

In another specific embodiment, the additional therapy is ananti-infective agent. Preferably, the anti-infective agent is one thatis not cytotoxic to healthy tissues surrounding and underlying the legulcer; thus, compounds such as iodine and bleach are disfavored. Thus,treatment of the leg ulcer, in one embodiment, comprises contacting theleg ulcer with collagen biofabric and administering an anti-infectiveagent. The anti-infective agent may be administered by any route, e.g.,topically, orally, buccally, intravenously, intramuscularly, anally,etc. In a specific example, the anti-infective agent is an antibiotic, abacteriostatic agent, antiviral compound, a virustatic agent, antifungalcompound, a fungistatic agent, or an antimicrobial compound. In anotherspecific embodiment, the anti-infective agent is ionic silver. In a morespecific embodiment, the ionic silver is contained within a hydrogel. Ina preferred embodiment, the collagen biofabric is impregnated withsilver ions prior to application to the leg ulcer. In anotherembodiment, the collagen biofabric is impregnated with silver ions afterapplication of the biofabric to the leg ulcer. In specific embodiments,the leg ulcer is a venous leg ulcer, arterial leg ulcer, decubitusulcer, or diabetic ulcer.

The invention further provides a method of treating a leg ulcercomprising contacting the leg ulcer with collagen biofabric and aplurality of stem or progenitor cells. In one embodiment, the collagenbiofabric may be contacted with the stem or progenitor cells prior tocontacting the leg ulcer with the collagen biofabric. For example, asheet or piece of collagen biofabric may be prepared immediately priorto application on the venous leg ulcer by disposing on the surface ofthe collagen biofabric a solution of stem or progenitor cells andallowing the stem or progenitor cells sufficient time to attach to thecollagen biofabric. The stem or progenitor cells, alternately, may bedisposed onto the surface of the collagen biofabric about 30 minutes, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24 or more hoursprior to application of the collagen biofabric onto the leg ulcer. Inanother embodiment, the collagen biofabric may be contacted with thestem or progenitor cells after application of the collagen biofabric tothe leg ulcer. In another embodiment, a the invention provides a methodof treating a venous leg ulcer comprising contacting the leg ulcer witha plurality of stem or progenitor cells, and contacting the leg ulcerwith collagen biofabric co that the collagen biofabric covers the legulcer and stem or progenitor cells.

The number of stem or progenitor cells disposed onto the leg ulcer, oronto the surface of the collagen biofabric, in any embodiment may vary,but may be at least 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹,3×10⁹, 1×10¹⁰, 3×10¹⁰, 1×10¹¹, 3×10¹¹ , or 1×10¹²; or may be no morethan 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹, 1×10¹⁰,3×10¹⁰, 1×10¹¹, 3×10¹¹, or 1×10¹² stem or progenitor cells. Thus, inspecific embodiments, the invention provides a method of treating a legulcer comprising contacting said leg ulcer with, in either order, (a)collagen biofabric, and (b) a plurality of stem or progenitor cellscomprising 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹,1×10¹⁰, 3×10¹⁰, 1×10¹¹, 3×10¹¹, or 1×10¹²; stem or progenitor cells. Inother specific embodiments, the invention provides a method of treatinga leg ulcer, in particular, a venous leg ulcer comprising contactingsaid leg ulcer with, in either order, (a) collagen biofabric, and (b) aplurality of stem or progenitor cells comprising no more than 1×10⁶,3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹, 1×10¹⁰, 3×10¹⁰, 1×10¹¹,3×10¹¹ , or 1×10¹²; stem or progenitor cells. In a more specificembodiment, said plurality of stem cells comprises two or more differentstem or progenitor cell types.

In one aspect, the invention provides a method of treating a leg ulcer,comprising contacting the leg ulcer with a collagen biofabric, whereinsaid collagen biofabric does not raise a detectable immune response tothe collagen biofabric. In a specific embodiment, the leg ulcer is avenous leg ulcer, arterial leg ulcer, decubitus ulcer, or diabeticulcer.

4.2 Collagen Biofabric

4.2.1 Description

The collagen biofabric used to treat a leg ulcer may be derived from theamniotic membrane of any mammal, for example, equine, bovine, porcine orcatarrhine sources, but is most preferably derived from human placenta.In a preferred embodiment, the collagen biofabric is substantially dry,i.e., is 20% or less water by weight. In another preferred embodiment,the collagen biofabric retains the native tertiary and quaternarystructure of the amniotic membrane, i.e., has not been protease-treated.In another preferred embodiment, the collagen biofabric contains nocollagen and other structural proteins that have been artificiallycrosslinked, e.g., chemically crosslinked, that is, the preferredcollagen biofabric is not fixed. A preferred collagen biofabric is thedried, non-fixed, non-protease-treated amniotic membrane materialdescribed in Hariri, U.S. Application Publication U.S. 2004/0048796,which is hereby incorporated in its entirety, and that is produced bythe methods described therein, and herein (see Examples 1, 2). However,the methods of the present invention can utilize any placenta-derivedcollagen material made by any procedure.

In a preferred embodiment, the collagen biofabric used in the treatmentof a leg ulcer is translucent. In other embodiments, the collagenbiofabric is opaque, or is colored or dyed, e.g., permanently colored ordyed, using a medically-acceptable dyeing or coloring agent; such anagent may be adsorbed onto the collagen biofabric, or the collagenbiofabric may be impregnated or coated with such an agent. In thisembodiment, any known non-toxic, non-irritating coloring agent or dyemay be used.

When the collagen biofabric is substantially dry, it is about 0.1 g/cm²to about 0.6 g/cm². In a specific embodiment, a single layer of thecollagen biofabric is at least 2 microns in thickness. In anotherspecific embodiment, a single layer of the collagen biofabric used torepair a tympanic membrane is approximately 10-40 microns in thickness,but may be approximately 2-150, 2-100 microns, 5-75 microns or 7-60microns in thickness in the dry state.

In one embodiment, the collagen biofabric is principally composed ofcollagen (types I, III and IV; about 90% of the matrix of thebiofabric), fibrin, fibronectin, elastin, and further containsglycosaminoglycans and proteoglycans. In other embodiments,non-structural components of the biofabric may include, for example,growth factors, e.g., platelet-derived growth factors (PDGFs),vascular-endothelial growth factor (VEGF), fibroblast growth factor(FGF) and transforming growth factor-β1. The composition of the collagenbiofabric is thus ideally suited to encourage the migration offibroblasts and macrophages, and thus the promotion of wound healing.

The collagen biofabric may be used in a single-layered format, forexample, as a single-layer sheet or an un-laminated membrane.Alternatively, the collagen biofabric may be used in a double-layer ormultiple-layer format, e.g., the collagen biofabric may be laminated.Lamination can provide greater stiffness and durability during thehealing process. The collagen biofabric may be, for example, laminatedas described below (see Section 4.2.7).

The collagen biofabric may further comprise collagen from a non-placentasource. For example, one or more layers of collagen biofabric may becoated or impregnated with, or layered with, purified extractedcollagen. Such collagen may be obtained, for example, from commercialsources, or may be produced according to known methods, such as thosedisclosed in U.S. Pat. Nos. 4,420,339, 5,814,328, and 5,436,135, thedisclosures of which are hereby incorporated by reference.

The collagen biofabric used to treat a leg ulcer may comprise one ormore compounds or substances that are not present in the placentalmaterial from which the collagen biofabric is derived. For example, thecollagen biofabric may be impregnated with a bioactive compound. Suchbioactive compounds include, but are not limited to, small organicmolecules (e.g., drugs), antibiotics (such as Clindamycin, Minocycline,Doxycycline, Gentamycin), hormones, growth factors, anti-tumor agents,anti-fungal agents, anti-viral agents, pain medications,anti-histamines, anti-inflammatory agents, anti-infectives including butnot limited to silver (such as silver salts, including but not limitedto silver nitrate and silver sulfadiazine), elemental silver,antibiotics, bactericidal enzymes (such as lysozyme), wound healingagents (such as cytokines including but not limited to PDGF, TGF;thymosin), hyaluronic acid as a wound healing agent, wound sealants(such as fibrin with or without thrombin), cellular attractant andscaffolding reagents (such as added fibronectin) and the like. In aspecific example, the collagen biofabric may be impregnated with atleast one growth factor, for example, fibroblast growth factor,epithelial growth factor, etc. The biofabric may also be impregnatedwith small organic molecules such as specific inhibitors of particularbiochemical processes e.g., membrane receptor inhibitors, kinaseinhibitors, growth inhibitors, anticancer drugs, antibiotics, etc.Impregnating the collagen biofabric with a bioactive compound may beaccomplished, e.g., by immersing the collagen biofabric in a solution ofthe bioactive compound of the desired concentration for a timesufficient to allow the collagen biofabric to absorb and to equilibratewith the solution; by spraying the solution onto the biofabric; bywetting the biofabric with the solution, etc..

In other embodiments, the collagen biofabric may be combined with ahydrogel. Any hydrogel composition known to one skilled in the art isencompassed within the invention, e.g., any of the hydrogel compositionsdisclosed in the following reviews: Graham, 1998, Med. Device Technol.9(1): 18-22; Peppas et al., 2000, Eur. J. Pharm. Biopharm. 50(1): 27-46;Nguyen et al., 2002, Biomaterials, 23(22): 4307-14; Henincl et al.,2002, Adv. Drug Deliv. Rev 54(1): 13-36; Skelhorne et al., 2002, Med.Device. Technol. 13(9): 19-23; Schmedlen et al., 2002, Biomaterials 23:4325-32; all of which are incorporated herein by reference in theirentirety. In a specific embodiment, the hydrogel composition is appliedon the collagen biofabric, i.e., disposed on the surface of the collagenbiofabric. The hydrogel composition for example, may be sprayed onto thecollagen biofabric or coated onto the surface of the collagen biofabric,or the biofabric may be soaked, bathed or saturated with the hydrogelcomposition. In another specific embodiment, the hydrogel is sandwichedbetween two or more layers of collagen biofabric. In an even morespecific embodiment, the hydrogel is sandwiched between two or morelayers of collagen biofabric, wherein the edges of the two layers ofbiofabric are sealed so as to substantially or completely contain thehydrogel.

The hydrogels useful in the methods and compositions of the inventioncan be made from any water-interactive, or water soluble polymer knownin the art, including but not limited to, polyvinylalcohol (PVA),polyhydroxyehthyl methacrylate, polyethylene glycol, polyvinylpyrrolidone, hyaluronic acid, alginate, collagen, gelatin, dextran orderivatives and analogs thereof.

In some embodiments, the collagen biofabric of the invention comprisesone or more bioactive compounds and is combined with a hydrogel. Forexample, the collagen biofabric can be impregnated with one or morebioactive compounds prior to being combined with a hydrogel. In otherembodiments, the hydrogel composition is further impregnated with one ormore bioactive compounds prior to, or after, being combined with acollagen biofabric of the invention, for example, the bioactivecompounds described in Section 4.2.2, below.

4.2.2 Bioactive Compounds

The collagen biofabric used in the methods of the invention may comprise(e.g., be impregnated with or coated with) one or more bioactivecompounds. As used herein, the term “bioactive compound” means anycompound or molecule that causes a measurable effect on one or morebiological systems in vitro or in vivo. Examples of bioactive compoundsinclude, without limitation, small organic molecules (e.g., drugs),minerals, metals, antibiotics, antiviral agents, antimicrobial agents,anti-inflammatory agents, antiproliferative agents, cytokines, enzyme orprotein inhibitors, antihistamines, and the like. In variousembodiments, the collagen biofabric may be coated or impregnated withantibiotics (such as Clindamycin, Minocycline, Doxycycline, Gentamycin),hormones, growth factors, anti-tumor agents, anti-fungal agents,anti-viral agents, pain medications (including Xylocaine®, Lidocaine,Procaine, Novocaine, etc.), antihistamines (e.g., diphenhydramine,Benadryl®, etc.), anti-inflammatory agents, anti-infectives includingbut not limited to silver (such as silver salts, including but notlimited to silver nitrate and silver sulfadiazine), elemental silver,antibiotics, bactericidal enzymes (such as lysozome), wound healingagents (such as cytokines including but not limited to PDGF (e.g.,Regranex®), TGF; thymosin), lactoferrin, hyaluronic acid as a woundhealing agent, wound sealants (such as fibrin with or without thrombin),cellular attractant and scaffolding reagents (such as fibronectin), andthe like, or combinations of any of the foregoing, or of the foregoingand other compounds not listed. Such impregnation or coating may beaccomplished by any means known in the art, and a portion or the wholeof the collagen biofabric may be so coated or impregnated.

The collagen biofabric, or composites comprising collagen biofabric, maycomprise any of the compounds listed herein, without limitation,individually or in any combination. Any of the biologically activecompounds listed herein, and others useful in the context of the scleraor eye, may be formulated by known methods for immediate release orextended release. Additionally, the collagen biofabric may comprise twoor more biologically active compounds in different manners; e.g., thebiofabric may be impregnated with one biologically active compound andcoated with another. In another embodiment, the collagen biofabriccomprises one biologically active compound formulated for extendedrelease, and a second biologically active compound formulated forimmediate release.

Wound healing, including the healing of leg ulcers, for example venousleg ulcers, requires adequate nutrition, particularly the presence ofiron, zinc, arginine, vitamin C, arginine, and the like. Thus, thecollagen biofabric may be impregnated or coated with aphysiologically-available form of one or more nutrients required forwound healing. Preferably, the nutrient is formulated for extendedrelease.

The collagen biofabric, or composite comprising collagen biofabric, maycomprise an antibiotic. In certain embodiments, the antibiotic is amacrolide (e.g., tobramycin (Tobi®)), a cephalosporin (e.g., cephalexin(Keflex®)), cephradine (Velosef®)), cefuroxime (Ceftin®, cefprozil(Cefzil®), cefaclor (Ceclor®), cefixime (Suprax® or cefadroxil(Duricef®), a clarithromycin (e.g., clarithromycin (Biaxin)), anerythromycin (e.g., erythromycin (EMycin®)), a penicillin (e.g.,penicillin V (V-CillinK® or Pen VeeK®)) or a quinolone (e.g., ofloxacin(Floxin®), ciprofloxacin (Cipro®) ornorfloxacin (Noroxin®)),aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins,butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin,paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicolantibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, andthiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin),carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem andimipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, andcefpirome), cephamycins (e.g., cefbuperazone, cefinetazole, andcefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,penicillin o-benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), macrolides (e.g., azithromycin, carbomycin, clarithomycin,dirithromycin, erythromycin, and erythromycin acistrate), amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin,tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, anddemeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans(e.g., furaltadone, and furazolium chloride), quinolones and analogsthereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, andgrepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), cycloserine, mupirocin andtuberin.

In certain embodiments, the collagen biofabric may be coated orimpregnated with an antifungal agent. Suitable antifungal agents includebut are not limited to amphotericin B, itraconazole, ketoconazole,fluconazole, intrathecal, flucytosine, miconazole, butoconazole,clotrimazole, nystatin, terconazole, tioconazole, ciclopirox, econazole,haloprogrin, naftifine, terbinafine, undecylenate, and griseofuldin.

In certain other embodiments, the collagen biofabric, or a compositecomprising collagen biofabric, is coated or impregnated with ananti-inflammatory agent. Useful anti-inflammatory agents include, butare not limited to, non-steroidal anti-inflammatory drugs such assalicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal,salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin,sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin,ketorolac, dichlofenac, ibuprofen, naproxen, naproxen sodium,fenoprofen, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam,ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone,oxyphenbutazone, antipyrine, aminopyrine, apazone and nimesulide;leukotriene antagonists including, but not limited to, zileuton,aurothioglucose, gold sodium thiomalate and auranofin; and otheranti-inflammatory agents including, but not limited to, methotrexate,colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarone.

In certain embodiments, the collagen biofabric, or a compositecomprising collagen biofabric, is coated or impregnated with anantiviral agent. Useful antiviral agents include, but are not limitedto, nucleoside analogs, such as zidovudine, acyclovir, gangcyclovir,vidarabine, idoxuridine, trifluridine, and ribavirin, as well asfoscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir,and the alpha-interferons.

The collagen biofabric, or a composite comprising collagen biofabric,may also be coated or impregnated with a cytokine receptor modulator.Examples of cytokine receptor modulators include, but are not limitedto, soluble cytokine receptors (e.g., the extracellular domain of aTNF-α receptor or a fragment thereof, the extracellular domain of anIL-10 receptor or a fragment thereof, and the extracellular domain of anIL-6 receptor or a fragment thereof), cytokines or fragments thereof(e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β, interferon (IFN)-α, IFN-β,IFN-γ, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFNreceptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax(Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12receptor antibodies), anti-cytokine antibodies (e.g., anti-IFNantibodies, anti-TNF-α antibodies, anti-IL-10 antibodies, anti-IL-6antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), andanti-IL-12 antibodies). In a specific embodiment, a cytokine receptormodulator is IL-4, IL-10, or a fragment thereof. In another embodiment,a cytokine receptor modulator is an anti-IL-1 antibody, anti-IL-6antibody, anti-IL-12 receptor antibody, or anti-TNF-α antibody. Inanother embodiment, a cytokine receptor modulator is the extracellulardomain of a TNF-α receptor or a fragment thereof. In certainembodiments, a cytokine receptor modulator is not a TNF-α antagonist.

In a preferred embodiment, proteins, polypeptides or peptides (includingantibodies) that are utilized as immunomodulatory agents are derivedfrom the same species as the recipient of the proteins, polypeptides orpeptides so as to reduce the likelihood of an immune response to thoseproteins, polypeptides or peptides. In another preferred embodiment,when the subject is a human, the proteins, polypeptides, or peptidesthat are utilized as immunomodulatory agents are human or humanized.

The collagen biofabric, or a composite comprising collagen biofabric,may also be coated or impregnated with a cytokine Examples of cytokinesinclude, but are not limited to, colony stimulating factor 1 (CSF-1),interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12),interleukin 15 (IL-15), interleukin 18 (IL-18), insulin-like growthfactor 1 (IGF-1), platelet derived growth factor (PDGF), erythropoietin(Epo), epidermal growth factor (EGF), fibroblast growth factor (FGF)(basic or acidic), granulocyte macrophage stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), heparin binding epidermalgrowth factor (HEGF), macrophage colony stimulating factor (M-CSF),prolactin, and interferon (IFN), e.g., IFN-alpha, and IFN-gamma),lactoferrin, transforming growth factor alpha (TGF-α), TGFβ1, TGFβ2,tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF), etc.

The collagen biofabric may also be coated or impregnated with a hormone.Examples of hormones include, but are not limited to, luteinizinghormone releasing hormone (LHRH), growth hormone (GH), growth hormonereleasing hormone, ACTH, somatostatin, somatotropin, somatomedin,parathyroid hormone, hypothalamic releasing factors, insulin, glucagon,enkephalins, vasopressin, calcitonin, heparin, low molecular weightheparins, heparinoids, synthetic and natural opioids, insulin thyroidstimulating hormones, and endorphins Examples of β-interferons include,but are not limited to, interferon β 1-a and interferon β 1-b.

The collagen biofabric, or composite comprising collagen biofabric, mayalso be coated or impregnated with an alkylating agent. Examples ofalkylating agents include, but are not limited to nitrogen mustards,ethylenimines, methylmelamines, alkyl sulfonates, nitrosoureas,triazenes, mechlorethamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, hexamethylmelaine, thiotepa, busulfan, carmustine,streptozocin, dacarbazine and temozolomide.

The collagen biofabric, or a composite comprising collagen biofabric,may also be coated or impregnated with an immunomodulatory agent,including but not limited to methothrexate, leflunomide,cyclophosphamide, cyclosporine A, macrolide antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators. peptidemimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal,polyclonal, Fvs, ScFvs, Fab or F(ab)₂ fragments or epitope bindingfragments), nucleic acid molecules (e.g., antisense nucleic acidmolecules and triple helices), small molecules, organic compounds, andinorganic compounds. In particular, immunomodulatory agents include, butare not limited to, methothrexate, leflunomide, cyclophosphamide,cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics(e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids,steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators. Examples ofT cell receptor modulators include, but are not limited to, anti-T cellreceptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412(Boeringer), IDEC-CE9.Is (IDEC and SKB), mAB 4162W94, Orthoclone andOKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (ProductDesign Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40ligand monoclonal antibodies (e.g., IDEC-131(IDEC)), anti-CD52antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies, anti-CD1 1aantibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g.,IDEC-114) (IDEC))) and CTLA4-immunoglobulin. In a specific embodiment, aT cell receptor modulator is a CD2 antagonist. In other embodiments, a Tcell receptor modulator is not a CD2 antagonist. In another specificembodiment, a T cell receptor modulator is a CD2 binding molecule,preferably MEDI-507. In other embodiments, a T cell receptor modulatoris not a CD2 binding molecule. The collagen biofabric comprising one ormore such immunomodulatory agents is useful in treating, e.g.,autoimmune conditions such as lupus or psoriasis, and vasculiticlesions.

The collagen biofabric, or composite comprising collagen biofabric, mayalso be coated or impregnated with a class of immunomodulatory compoundsknown as IMiDs. As used herein and unless otherwise indicated, the term“IMiD®” and “IMiDs®” (Celgene Corporation) encompasses small organicmolecules that markedly inhibit TNF-α, LPS induced monocyte IL1β andIL12, and partially inhibit IL6 production. Specific immunomodulatorycompounds are discussed below.

Specific examples of immunomodulatory compounds include cyano andcarboxy derivatives of substituted styrenes such as those disclosed inU.S. Pat. No. 5,929,117; 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl)isoindolines and 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl)isoindolines such as those described in U.S. Pat. Nos. 5,874,448 and5,955,476; the tetra substituted2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines(e.g., 4-methyl derivatives of thalidomide), substituted2-(2,6-dioxopiperidin-3-yl) phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles including, but not limitedto, those disclosed in U.S. Pat. Nos. 5,635,517, 6,281,230, 6,316,471,6,403,613, 6,476,052 and 6,555,554; 1-oxo and 1,3-dioxoisoindolinessubstituted in the 4- or 5-position of the indoline ring (e.g.,4-(4-amino-1,3-dioxoisoindoline-2-yl)-4-carbamoylbutanoic acid)described in U.S. Pat. No. 6,380,239; isoindoline-1-one andisoindoline-1,3-dione substituted in the 2-position with2,6-dioxo-3-hydroxypiperidin-5-yl (e.g.,2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one)described in U.S. Pat. No. 6,458,810; a class of non-polypeptide cyclicamides disclosed in U.S. Pat. Nos. 5,698,579 and 5,877,200; andisoindole-imide compounds such as those described in U.S. patentpublication no. 2003/0045552 published on Mar. 6, 2003, U.S. patentpublication no. 2003/0096841 published on May 22, 2003, andInternational Application No. PCT/US01/50401 (International PublicationNo. WO 02/059106). The entireties of each of the patents and patentapplications identified herein are incorporated herein by reference.Immunomodulatory compounds do not include thalidomide.

Various immunomodulatory compounds contain one or more chiral centers,and can exist as racemic mixtures of enantiomers or mixtures ofdiastereomers. This invention encompasses the use of stereomericallypure forms of such compounds, as well as the use of mixtures of thoseforms. For example, mixtures comprising equal or unequal amounts of theenantiomers of a particular immunomodulatory compounds may be used inmethods and compositions. These isomers may be asymmetricallysynthesized or resolved using standard techniques such as chiral columnsor chiral resolving agents. See, e.g., Jacques, J., et al., Enantiomers,Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind., 1972).

Preferred immunomodulatory compounds include, but are not limited to,1-oxo- and 1,3 dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolinessubstituted with amino in the benzo ring as described in U.S. Pat. No.5,635,517 which is incorporated herein by reference. These compoundshave the structure I:

in which one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;

1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and

1,3-dioxo-2-(3-methyl-2,6-dioxopiperidin-3-yl)-4-aminoisoindole, andoptically pure isomers thereof. The compounds can be obtained viastandard, synthetic methods (see e.g., U.S. Pat. No. 5,635,517,incorporated herein by reference). The compounds are also available fromCelgene Corporation, Warren, N.J.

As used herein, and unless otherwise indicated, the term “opticallypure” means a composition that comprises one optical isomer of acompound and is substantially free of other isomers of that compound.For example, an optically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. An optically pure composition of a compound having twochiral centers will be substantially free of other diastereomers of thecompound. A typical optically pure compound comprises greater than about80% by weight of one enantiomer of the compound and less than about 20%by weight of other enantiomers of the compound, more preferably greaterthan about 90% by weight of one enantiomer of the compound and less thanabout 10% by weight of the other enantiomers of the compound, even morepreferably greater than about 95% by weight of one enantiomer of thecompound and less than about 5% by weight of the other enantiomers ofthe compound, more preferably greater than about 97% by weight of oneenantiomer of the compound and less than about 3% by weight of the otherenantiomers of the compound, and most preferably greater than about 99%by weight of one enantiomer of the compound and less than about 1% byweight of the other enantiomers of the compound.

Other specific immunomodulatory compounds belong to a class ofsubstituted 2-(2,6-dioxopiperidin-3-yl) phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as those described inU.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and 6,476,052, andInternational Patent Application No. PCT/US97/13375 (InternationalPublication No. WO 98/03502), each of which is incorporated herein byreference. Representative compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, and R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or halo;

-   -   provided that R⁶ is other than hydrogen if X and Y are C═O        and (i) each of R¹, R², R³, and R⁴ is fluoro or (ii) one of R¹,        R², R³, or R⁴ is amino.

Compounds representative of this class are of the formulas:

wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory compounds belong to a class ofisoindole-imides disclosed in U.S. Patent Application Publication Nos.US 2003/0096841 and US 2003/0045552, and International Application No.PCT/US01/50401 (International Publication No. WO 02/059106), each ofwhich are incorporated herein by reference. Representative compounds areof formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

-   -   n is 0 or 1; and

* represents a chiral-carbon center.

In specific compounds of formula II, when n is 0 then R¹ is(C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,C(O)R³, C(O)OR⁴, (C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, C(S)NHR³, or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H or (C₁-C₈)alkyl; and

R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

In other specific compounds of formula II, R² is H or (C₁-C₄)alkyl.

In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl or benzyl.

In other specific compounds of formula II, R¹ is H, (C₁-C₈)alkyl,benzyl, CH₂OCH₃, CH₂CH₂OCH₃, or

In another embodiment of the compounds of formula II, R¹ is

wherein Q is O or S, and each occurrence of R⁷ is independently H,(C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl,aryl, halogen, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, (C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵, or adjacentoccurrences of R⁷ can be taken together to form a bicyclic alkyl or arylring.

In other specific compounds of formula II, R¹ is C(O)R³.

In other specific compounds of formula II, R³ is(C0-C4)alkyl-(C2-C5)heteroaryl, (C1-C8)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

In other specific compounds of formula II, heteroaryl is pyridyl, furyl,or thienyl.

In other specific compounds of formula II, R¹ is C(O)OR⁴.

In other specific compounds of formula II, the H of C(O)NHC(O) can bereplaced with (C₁-C₄)alkyl, aryl, or benzyl.

Further examples of the compounds in this class include, but are notlimited to:[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide;(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamicacid tert-butyl ester;4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-acetamide;N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-carboxamide;2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridylcarboxamide;3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione;2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide;{N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methylacetate;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamino)carboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide;andN-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.

Still other specific immunomodulatory compounds belong to a class ofisoindole-imides disclosed in U.S. Patent Application Publication Nos.US 2002/0045643, International Publication No. WO 98/54170, and U.S.Pat. No. 6,395,754, each of which is incorporated herein by reference.Representative compounds are of formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center.

Other representative compounds are of formula:

wherein:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂ X¹CH₂CH₂— in which X¹ is —O—,—S—, or —NH—; and

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl.

Other representative compounds are of formula:

in which

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

each of R¹, R², R³, and R⁴, independently of the others, is halo, alkylof 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one ofR¹, R², R³, and R⁴ is nitro or protected amino and the remaining of R¹,R², R³, and R⁴ are hydrogen; and

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.

Other representative compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, and R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen, alkyl of 1 to 8 carbon atoms, or CO—R⁷—CH(R¹⁰)NR⁸R⁹ inwhich each of R⁷, R⁸, R⁹, and R¹⁰ is as herein defined; and

R⁶ is alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.

Specific examples of the compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, chloro, or fluoro;

R⁷ is m-phenylene, p-phenylene or —(C_(n)H_(2n))— in which n has a valueof 0 to 4; each of R⁸ and R⁹ taken independently of the other ishydrogen or alkyl of 1 to 8 carbon atoms, or R⁸ and R⁹ taken togetherare tetramethylene, pentamethylene, hexamethylene, or —CH₂CH₂X¹CH₂CH₂—in which X¹ is —O—, —S— or —NH—; and

R¹⁰ is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl.

Preferred immunomodulatory compounds are4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione has thefollowing chemical structure:

The compound3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione has thefollowing chemical structure:

In another embodiment, specific immunomodulatory compounds encompasspolymorphic forms of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione such as Form A, B, C, D, E,F, G and H, disclosed in U.S. provisional application No. 60/499,723filed on Sep. 4, 2003, and U.S. non-provisional application Ser. No.10/934,863, filed Sep. 3, 2004, both of which are incorporated herein byreference. For example, Form A of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated,crystalline material that can be obtained from non-aqueous solventsystems. Form A has an X-ray powder diffraction pattern comprisingsignificant peaks at approximately 8, 14.5, 16, 17.5, 20.5, 24 and 26degrees 2θ, and has a differential scanning calorimetry meltingtemperature maximum of about 270° C. Form A is weakly or not hygroscopicand appears to be the most thermodynamically stable anhydrous polymorphof 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dionediscovered thus far.

Form B of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a hemihydrated,crystalline material that can be obtained from various solvent systems,including, but not limited to, hexane, toluene, and water. Form B has anX-ray powder diffraction pattern comprising significant peaks atapproximately 16, 18, 22 and 27 degrees 2θ, and has endotherms from DSCcurve of about 146 and 268° C., which are identified dehydration andmelting by hot stage microscopy experiments. Interconversion studiesshow that Form B converts to Form E in aqueous solvent systems, andconverts to other forms in acetone and other anhydrous systems.

Form C of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a hemisolvatedcrystalline material that can be obtained from solvents such as, but notlimited to, acetone. Form C has an X-ray powder diffraction patterncomprising significant peaks at approximately 15.5 and 25 degrees 2θ,and has a differential scanning calorimetry melting temperature maximumof about 269° C. Form C is not hygroscopic below about 85% RH, but canconvert to Form B at higher relative humidities.

Form D of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a crystalline, solvatedpolymorph prepared from a mixture of acetonitrile and water. Form D hasan X-ray powder diffraction pattern comprising significant peaks atapproximately 27 and 28 degrees 2θ, and has a differential scanningcalorimetry melting temperature maximum of about 270° C. Form D iseither weakly or not hygroscopic, but will typically convert to Form Bwhen stressed at higher relative humidities.

Form E of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is a dihydrated, crystallinematerial that can be obtained by slurrying 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione in water and by a slowevaporation of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione in a solvent system with aratio of about 9:1 acetone:water. Form E has an X-ray powder diffractionpattern comprising significant peaks at approximately 20, 24.5 and 29degrees 2θ, and has a differential scanning calorimetry meltingtemperature maximum of about 269° C. Form E can convert to Form C in anacetone solvent system and to Form G in a THF solvent system. In aqueoussolvent systems, Form E appears to be the most stable form. Desolvationexperiments performed on Form E show that upon heating at about 125° C.for about five minutes, Form E can convert to Form B. Upon heating at175° C. for about five minutes, Form B can convert to Form F.

Form F of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated,crystalline material that can be obtained from the dehydration of FormE. Form F has an X-ray powder diffraction pattern comprising significantpeaks at approximately 19, 19.5 and 25 degrees 2θ, and has adifferential scanning calorimetry melting temperature maximum of about269° C.

Form G of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is an unsolvated,crystalline material that can be obtained from slurrying forms B and Ein a solvent such as, but not limited to, tetrahydrofuran (THF). Form Ghas an X-ray powder diffraction pattern comprising significant peaks atapproximately 21, 23 and 24.5 degrees 2θ, and has a differentialscanning calorimetry melting temperature maximum of about 267° C. Form Hof 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidene-2,6-dione is apartially hydrated (about 0.25 moles) crystalline material that can beobtained by exposing Form E to 0% relative humidity. Form H has an X-raypowder diffraction pattern comprising significant peaks at approximately15, 26 and 31 degrees 2θ, and has a differential scanning calorimetrymelting temperature maximum of about 269° C.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such asthose described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each of whichis incorporated herein by reference. Representative compounds are offormula:

wherein:

Y is oxygen or H² and

each of R¹, R², R³, and R⁴, independently of the others, is hydrogen,halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, oramino.

Other specific immunomodulatory compounds include, but are not limitedto, the tetra substituted 2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolinesdescribed in U.S. Pat. No. 5,798,368, which is incorporated herein byreference. Representative compounds are of formula:

wherein each of R¹, R², R³, and R⁴, independently of the others, ishalo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolinesdisclosed in U.S. Pat. No. 6,403,613, which is incorporated herein byreference. Representative compounds are of formula:

in which

Y is oxygen or H₂,

a first of R¹ and R² is halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, the second of R¹ and R², independently of thefirst, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, and

R³ is hydrogen, alkyl, or benzyl.

Specific examples of the compounds are of formula:

wherein

a first of R¹ and R² is halo, alkyl of from 1 to 4 carbon atoms, alkoxyof from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from1 to 4 carbon atoms, cyano, or carbamoyl;

the second of R¹ and R², independently of the first, is hydrogen, halo,alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms,alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylaminoin which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl;and

R³ is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl. Specificexamples include, but are not limited to,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

Other representative compounds are of formula:

wherein:

a first of R¹ and R² is halo, alkyl of from 1 to 4 carbon atoms, alkoxyof from 1 to 4 carbon atoms, dialkylamino in which each alkyl is of from1 to 4 carbon atoms, cyano, or carbamoyl;

the second of R¹ and R², independently of the first, is hydrogen, halo,alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms,alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylaminoin which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl;and

R³ is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or 5-positionof the indoline ring described in U.S. Pat. No. 6,380,239 and co-pendingU.S. application Ser. No. 10/900,270, filed Jul. 28, 2004, which areincorporated herein by reference. Representative compounds are offormula:

in which the carbon atom designated C* constitutes a center of chirality(when n is not zero and R¹ is not the same as R²); one of X¹ and X² isamino, nitro, alkyl of one to six carbons, or NH—Z, and the other of X¹or X² is hydrogen; each of R¹ and R² independent of the other, ishydroxy or NH—Z; R³ is hydrogen, alkyl of one to six carbons, halo, orhaloalkyl; Z is hydrogen, aryl, alkyl of one to six carbons, formyl, oracyl of one to six carbons; and n has a value of 0, 1, or 2; providedthat if X¹ is amino, and n is 1 or 2, then R¹ and R² are not bothhydroxy; and the salts thereof.

Further representative compounds are of formula:

in which the carbon atom designated C* constitutes a center of chiralitywhen n is not zero and R¹ is not R²; one of X¹ and X² is amino, nitro,alkyl of one to six carbons, or NH—Z, and the other of X¹ or X² ishydrogen; each of R¹ and R² independent of the other, is hydroxy orNH—Z; R³ is alkyl of one to six carbons, halo, or hydrogen; Z ishydrogen, aryl or an alkyl or acyl of one to six carbons; and n has avalue of 0, 1, or 2.

Specific examples include, but are not limited to,2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid and4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-cabamoyl-butyric acid,which have the following structures, respectively, and pharmaceuticallyacceptable salts, solvates, prodrugs, and stereoisomers thereof:

Other representative compounds are of formula:

in which the carbon atom designated C* constitutes a center of chiralitywhen n is not zero and R¹ is not R²; one of X¹ and X² is amino, nitro,alkyl of one to six carbons, or NH—Z, and the other of X¹ or X² ishydrogen; each of R¹ and R² independent of the other, is hydroxy orNH—Z; R³ is alkyl of one to six carbons, halo, or hydrogen; Z ishydrogen, aryl, or an alkyl or acyl of one to six carbons; and n has avalue of 0, 1, or 2; and the salts thereof

Specific examples include, but are not limited to,4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-phenylcarbamoyl-butyricacid, and2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pentanedioicacid, which have the following structures, respectively, andpharmaceutically acceptable salts, solvate, prodrugs, and stereoisomersthereof:

Other specific examples of the compounds are of formula:

wherein:

one of X¹ and X² is nitro, or NH—Z, and the other of X¹ or X² ishydrogen;

each of R¹ and R², independent of the other, is hydroxy or NH—Z;

R³ is alkyl of one to six carbons, halo, or hydrogen;

Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of oneto six carbons; and

n has a value of 0, 1, or 2; and

if —COR² and —(CH₂)_(n)COR¹ are different, the carbon atom designated C*constitutes a center of chirality.

Other representative compounds are of formula:

wherein:

one of X¹ and X² is alkyl of one to six carbons;

each of R¹ and R², independent of the other, is hydroxy or NH—Z;

R³ is alkyl of one to six carbons, halo, or hydrogen;

Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of oneto six carbons; and

n has a value of 0, 1, or 2; and

if —COR² and —(CH₂)_(n)COR¹ are different, the carbon atom designated C*constitutes a center of chirality.

Still other specific immunomodulatory compounds include, but are notlimited to, isoindoline-1-one and isoindoline-1,3-dione substituted inthe 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S.Pat. No. 6,458,810, which is incorporated herein by reference.Representative compounds are of formula:

wherein:

the carbon atoms designated * constitute centers of chirality;

X is —C(O)— or —CH₂—;

R¹ is alkyl of 1 to 8 carbon atoms or —NHR³;

R² is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen; and

R³ is hydrogen,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxyof 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbonatoms,

cycloalkyl of 3 to 18 carbon atoms,

phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms,

benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms, or —COR⁴ in which

R⁴ is hydrogen,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxyof 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbonatoms,

cycloalkyl of 3 to 18 carbon atoms,

phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms, or

benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms.

The immunomodulatory compounds disclosed herein can either becommercially purchased or prepared according to the methods described inthe patents or patent publications disclosed herein. Further, opticallypure compounds can be asymmetrically synthesized or resolved using knownresolving agents or chiral columns as well as other standard syntheticorganic chemistry techniques.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein, and unless otherwise specified, the term “solvate” meansa compound of the present invention or a salt thereof, that furtherincludes a stoichiometric or non-stoichiometric amount of solvent boundby non-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of immunomodulatory compounds that comprise biohydrolyzablemoieties such as biohydrolyzable amides, biohydrolyzable esters,biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzableureides, and biohydrolyzable phosphate analogues. Other examples ofprodrugs include derivatives of immunomodulatory compounds that compriseNO, NO2, ONO, or ONO2 moieties. Prodrugs can typically be prepared usingwell known methods, such as those described in 1 Burger's MedicinalChemistry and Drug Discovery, 172 178, 949 982 (Manfred E. Wolff ed.,5th ed. 1995), and Design of Prodrugs (H. Bundgaard ed., Elselvier, NewYork 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyl-oxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, a amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, amino acids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

As used herein, and unless otherwise specified, the term “stereoisomer”encompasses all enantiomerically/stereomerically pure andenantiomerically/stereomerically enriched compounds of this invention.

As used herein, and unless otherwise indicated, the term“stereomerically pure” or “enantiomerically pure” means that a compoundcomprises one stereoisomer and is substantially free of its counterstereoisomer or enantiomer. For example, a compound is stereomericallyor enantiomerically pure when the compound contains 80%, 90%, or 95% ormore of one stereoisomer and 20%, 10%, or 5% or less of the counterstereoisomer. In certain cases, a compound is considered opticallyactive or stereomerically/enantiomerically pure (i.e., substantially theR-form or substantially the S-form) with respect to a chiral center whenthe compound is about 80% ee (enantiomeric excess) or greater,preferably, equal to or greater than 90% ee with respect to a particularchiral center, and more preferably 95% ee with respect to a particularchiral center.

As used herein, and unless otherwise indicated, the term“stereomerically enriched” or “enantiomerically enriched” encompassesracemic mixtures as well as other mixtures of stereoisomers of compoundsof this invention (e.g., R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40,65/35 and 70/30). Various immunomodulatory compounds of the inventioncontain one or more chiral centers, and can exist as racemic mixtures ofenantiomers or mixtures of diastereomers. This invention encompasses theuse of stereomerically pure forms of such compounds, as well as the useof mixtures of those forms. For example, mixtures comprising equal orunequal amounts of the enantiomers of a particular immunomodulatorycompounds of the invention may be used in methods and compositions ofthe invention. These isomers may be asymmetrically synthesized orresolved using standard techniques such as chiral columns or chiralresolving agents. See, e.g., Jacques, J., et al., Enantiomers, Racematesand Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., etal., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of CarbonCompounds (McGraw Hill, NY, 1962); and Wilen, S. H., Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind., 1972).

Compounds used in the invention may be small organic molecules having amolecular weight less than about 1,000 g/mol, and are not proteins,peptides, oligonucleotides, oligosaccharides or other macromolecules.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

The amount of the bioactive compound coating or impregnating thecollagen may vary, and will preferably depend upon the particularbioactive compound to be delivered, and the effect desired. For example,where the bioactive compound is an anti-inflammatory agent, the amountof the anti-inflammatory agent on or contained by the collagen is anamount sufficient to measurably reduce one or more symptoms or indiciaof inflammation in or around the leg ulcer.

In various embodiments, the collagen biofabric, used to treat a legulcer, may be coated with, or impregnated with, at least 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800,900, 100, 1250, 1500, 2000, 2500, 300, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000,40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000 or at least 1000000nanograms of a bioactive compound. In another embodiment, the ocularplug of the invention may be coated with, or impregnated with, no morethan 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400,500, 600, 700, 800, 900, 100, 1250, 1500, 2000, 2500, 300, 3500, 4000,4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000,300000, 400000, 500000, 600000, 700000, 800000, 900000 or at least1000000 nanograms of a bioactive compound.

4.2.3 Conformation of the Collagen Biofabric

The collagen biofabric may be formed into any shape or conformation thatwill facilitate its use in the methods of the invention. for example,the collagen biofabric can be formed in any shape or conformation thatwill facilitate the healing of a leg ulcer, e.g., a venous leg ulcer.For example, the collagen biofabric may be cut to various sizes so as tocover typical leg ulcers completely. The collagen biofabric may also beprovided in various sizes so as to enable a physician, or other enduser, to use or to cut an appropriately-sized piece for treatment of aleg ulcer, e.g., a venous leg ulcer. The collagen biofabric may be cutto a square, rectangular or oval shaped pieces, for example, or may becut to conform to the shape of a particular ulcer on a particularpatient. In various embodiments of the method, collagen biofabric piecesused to treat a leg ulcer, particularly a venous leg ulcer, may beapproximately 1×1 cm, 1.5×1.5 cm, 2×2 cm, 2.5×2.5 cm, 3×3 cm, 3.5×3.5cm, 4×4 cm, 4.5×4.5 cm, 5×5 cm, 1×1.5 cm, 1×2 cm, 1×2.5 cm, 1×3 cm,1×3.5 cm, 1×4 cm, 1×4.5 cm, 1×5 cm, 1.5×2 cm, 1.5×2.5 cm, 1.5×3 cm,1.5×3.5 cm, 1.5×4 cm, 1.5×4.5 cm, 2×2.5 cm, 2×3 cm, 2×3.5 cm, 2×4 cm,2×4.5 cm, 2×5 cm, 2.5×3 cm, 2.5×3.5 cm, 2.5×4 cm, 2.5×4.5 cm, 2.5×5 cm,3×3.5 cm, 3×4 cm, 3×4.5 cm, 3×5 cm, 3.5×4 cm, 3.5×4.5 cm, 3.5×5 cm,4×4.5 cm, 4×5 cm, 4.5×5 cm in size, or up to 6×8 cm in size, or may beno smaller, or no larger, than 1×1 cm, 1.5×1.5 cm, 2×2 cm, 2.5×2.5 cm,3×3 cm, 3.5×3.5 cm, 4×4 cm, 4.5×4.5 cm, 5×5 cm, 1×1.5 cm, 1×2 cm, 1×2.5cm, 1×3 cm, 1×3.5 cm, 1×4 cm, 1×4.5 cm, 1×5 cm, 1.5×2 cm, 1.5×2.5 cm,1.5×3 cm, 1.5×3.5 cm, 1.5×4 cm, 1.5×4.5 cm, 2×2.5 cm, 2×3 cm, 2×3.5 cm,2×4 cm, 2×4.5 cm, 2×5 cm, 2.5×3 cm, 2.5×3.5 cm, 2.5×4 cm, 2.5×4.5 cm,2.5×5 cm, 3×3.5 cm, 3×4 cm, 3×4.5 cm, 3×5 cm, 3.5×4 cm, 3.5×4.5 cm,3.5×5 cm, 4×4.5 cm, 4×5 cm, or 4.5×5 cm, though the biofabric may be cutto different dimensions. Further, the biofabric used to treat a legulcer, particularly a venous leg ulcer, may be provided as a sheet fromwhich an end use may cut two or more pieces, or may be provided as aroll or strip.

In one embodiment, the collagen biofabric is provided in a single sheetto be laid whole on a leg ulcer. In another embodiment, the collagenbiofabric is provided as a mesh, netting or webbing that is capable ofbeing spread out across the leg ulcer. In this embodiment, the collagenbiofabric does not need to completely cover the surface area of the legulcer, but facilitates ingrowth of epithelial tissue along, and adjacentto, the collagen biofabric. Such a mesh, netting or webbing may beprovided, in one embodiment, as a sheet of collagen biofabric containinga plurality of cuts enabling the end user to spread the sheet out into anetting, webbing or mesh. Such a mesh, netting or webbing may be madefrom a sheet of collagen biofabric by, for example, stamping, cutting orslicing, either manually or using a machine. Preferably, the stamping,cutting or slicing is performed under sterile conditions.

The collagen biofabric useful in the treatment methods of the inventionmay be provided to the end user either dry, or pre-wetted in a suitablephysiologically-compatible, medically-useful liquid, such as a salinesolution. In one embodiment, the solution comprises one or morebioactive compounds, as described in Section 4.2.2, above, withoutlimitation. Preferably, said bioactive compound is disposed onto orwithin the collagen biofabric such that the majority of the bioactivecompound contacts the leg ulcer during the time the collagen biofabriccontacts the leg ulcer.

In another embodiment, the collagen biofabric is layered onto a support.Such a support may be non-natural, such as a plastic sheet or film, or acloth woven of non-natural fibers, or may be natural, such as a clothwoven of natural fibers, or a non-collagen biofabric dermal replacement.In one embodiment, the support is a bandage; in this embodiment, thebandage and the collagen biofabric form a layered wound covering, withthe bandage holding the collagen biofabric against the leg ulcer. Inanother embodiment, a sheet of collagen biofabric comprises a compoundor material that is able to absorb excess wound exudates. In a specificembodiment, the compound or material is a collagen gel. In a morespecific embodiment, said collagen gel is disposed on the fetal side ofthe collagen biofabric. In another more specific embodiment, saidcollagen gel is disposed on the maternal side of the collagen biofabric.In another more specific embodiment, said collagen gel is disposed onboth sides of the collagen biofabric.

4.2.4 Method of Making Collagen Biofabric

Collagen biofabric, made from amniotic membrane, chorionic membrane, orboth, may be produced by any means that preserves the biochemical andstructural characteristics of the membrane's components—chieflycollagen, elastin, laminin, and fibronectin. A preferred material is thecollagen biofabric described in, and produced according to the methodsdisclosed in, United States Application Publication No. U.S.2004/0048796 A1, “Collagen Biofabric and Methods of Preparation and UseTherefor” by Hariri, which is hereby incorporated herein in itsentirety.

Preferably, the collagen biofabric used to treat a leg ulcer is from ahuman placenta for use in human subjects, though the collagen biofabricmay be made from amniotic membrane from a non-human mammal. Where thecollagen biofabric is to be used in a non-human animal, it is preferredthat the collagen biofabric be derived from a placenta from that speciesof animal

In a preferred embodiment, the placenta for use in the methods of theinvention is taken as soon as possible after delivery of the newborn.The placenta may be used immediately, or may be stored for 2-5 days fromthe time of delivery prior to any further treatment. The placenta istypically exsanguinated, that is, drained of the cord blood remainingafter birth. Preferably, the expectant mother is screened prior to thetime of birth, using standard techniques known to one skilled in theart, for communicable diseases including but not limited to, HIV, HBV,HCV, HTLV, syphilis, CMV, and other viral pathogens known to contaminateplacental tissue.

One exemplary method for preparing a collagen biofabric of the inventioncomprises the following steps:

Step I.

The umbilical cord is separated from the placental disc; optionally, theamniotic membrane is separated from the chorionic membrane. In apreferred embodiment, the amniotic membrane is separated from thechorionic membrane prior to cutting the placental membrane. Followingseparation of the amniotic membrane from the chorionic membrane andplacental disc, the umbilical cord stump is cut, e.g., with scissors,and detached from the placental disc. The amniotic membrane may then bestored in a sterile, preferably buffered, saline solution, such as 0.9%sterile NaCl solution. Preferably, the amniotic membrane is stored byrefrigeration, at a temperature of at least 2° C.

Step II.

The amniotic membrane is substantially decellularized; that is,substantially all cellular material and cellular debris (e.g., allvisible cellular material and cellular debris) is removed. Anydecellularizing process known to one skilled in the art may be used,however, generally the process used for decellularizing the amnioticmembrane of the invention does not disrupt the native conformation ofthe proteins making up the biofabric. “Substantial decellularization” ofthe amniotic membrane preferably removes at least 90% of the cells, morepreferably removes at least 95% of the cells, and most preferablyremoves at least 99% of the cells (e.g., fibroblasts, amniocytes andchorionocytes). The amniotic membranes decellularized in accordance withthe methods of the invention are uniformly thin, with inherent thicknessvariations of between about 2 and about 150 microns in the dry state,smooth (as determined by touch) and clear in appearance.Decellularization may comprise physical scraping, for example, with asterile cell scraper, in combination with rinsing with a sterilesolution. The decellularization technique employed should not result ingross disruption of the anatomy of the amniotic membrane or alter thebiomechanical properties of the amniotic membrane. Preferably, thedecellularization of the amniotic membrane comprises use of adetergent-containing solution, such as nonionic detergents, TritonX-100, anionic detergents, sodium dodecyl sulfate, Any mild anionicdetergent, i.e., a non-caustic detergent, with a pH of 6 to 8, and lowfoaming, can be used to decellularize the amniotic membrane. In aspecific embodiment, 0.01-1% deoxycholic acid sodium salt monohydrate isused in the decellularization of the amniotic membrane.

It is highly preferable to limit the protease activity in preparation ofthe biofabric. Additives to the lysis, rinse and storage solutions suchas metal ion chelators, for example 1,10-phenanthroline andethylenediaminetetraacetic acid (EDTA), create an environmentunfavorable to many proteolytic enzymes. Providing sub-optimalconditions for proteases such as collagenase, assists in protectingamniotic membrane components such as collagen from degradation duringthe cell lysis step. Suboptimal conditions for proteases may be achievedby formulating the hypotonic lysis solution to eliminate or limit theamount of calcium and zinc ions available in solution. Many proteasesare active in the presence of calcium and zinc ions and lose much oftheir activity in calcium and zinc ion free environments. Preferably,the hypotonic lysis solution will be prepared selecting conditions ofpH, reduced availability of calcium and zinc ions, presence of metal ionchelators and the use of proteolytic inhibitors specific for collagenasesuch that the solution will optimally lyse the native cells whileprotecting the underlying amniotic membrane from adverse proteolyticdegradation. For example a hypotonic lysis solution may include abuffered solution of water, pH 5.5 to 8, preferably pH 7 to 8, free fromcalcium and zinc ions and including a metal ion chelator such as EDTA.Additionally, control of the temperature and time parameters during thetreatment of the amniotic membrane with the hypotonic lysis solution mayalso be employed to limit the activity of proteases.

It is preferred that the decellularization treatment of the amnioticmembrane also limits the generation of new immunological sites. Sinceenzymatic degradation of collagen is believed to lead to heightenedimmunogenicity, the invention encompasses treatment of the amnioticmembrane with enzymes, e.g., nucleases, that are effective in inhibitingcellular metabolism, protein production and cell division, that minimizeproteolysis of the compositions of the amniotic membrane thus preservingthe underlying architecture of the amniotic membrane. Examples ofnucleases that can be used in accordance with the methods of theinvention are those effective in digestion of native cell DNA and RNAincluding both exonucleases and endonucleases. A non-limiting example ofnucleases that can be used in accordance with the methods of theinvention include exonucleases that inhibit cellular activity, e.g.,DNase I (SIGMA Chemical Company, St. Louis, Mo.) and RNase A (SIGMAChemical Company, St. Louis, Mo.) and endonucleases that inhibitcellular activity, e.g., EcoRI (SIGMA Chemical Company, St. Louis, Mo.)and HindIll (SIGMA Chemical Company, St. Louis, Mo.). It is preferablethat the selected nucleases are applied in a physiological buffersolution which contains ions, e.g., magnesium, calcium, which areoptimal for the activity of the nuclease. Preferably, the ionicconcentration of the buffered solution, the treatment temperature andthe length of treatment are selected by one skilled in the art byroutine experimentation to assure the desired level of nucleaseactivity. The buffer is preferably hypotonic to promote access of thenucleases to cell interiors.

In another embodiment of Steps I and II, above, the placenta, afterinitial processing, is briefly rinsed in saline to remove blood from theplacental surface. The placental disk is then immersed in a colddeoxycholic acid solution at a concentration of about 0.1% to about 10%,and, in a specific embodiment, about 0.1% to about 2.0%. The placenta isthen incubated in this solution at between about 1° C. to about 8° C.for about 5 days to about 6 months. In specific embodiments, theplacental disk is immersed, for example, for about 5 to about 15 days;about 5 to about 30 days, about 5 to about 60 days, or for up to aboutone year. Typically, the deoxycholic acid solution is replaced duringincubation every 2-5 days. In another specific embodiment, the placentaldisk is immersed in a deoxycholic acid solution at a concentration ofabout 1% at a temperature of 0° C. to about 8° C. for about 5 days toabout 15 days. This incubation serves two purposes. First, it allowstime for serological tests to be performed on the placental material andblood, so that placentas failing to meet serological criteria are notprocessed further. Second, the longer incubation improves the removal ofepithelial cells and fibroblasts, which allows for a significantreduction in the amount of time spent decellularizing the amnion byphysically scraping. Typically, the scraping time is reduced from, e.g.,about 40 minutes to about 20 minutes. The amniotic membrane is thendried as described below.

Step III.

Following decellularization, the amniotic membrane is washed to assureremoval of cellular debris which may include cellular proteins, cellularlipids, and cellular nucleic acids, as well as any extracellular debrissuch as extracellular soluble proteins, lipids and proteoglycans. Thewash solution may be de-ionized water or an aqueous hypotonic buffer.Preferably, the amniotic membrane is gently agitated for 15-120 minutesin the detergent, e.g., on a rocking platform, to assist in thedecellularization. The amniotic membrane may, after detergentdecellularization, again be physically decellularized as describedabove; the physical and detergent decellularization steps may berepeated as necessary, as long as the integrity of the amniotic membraneis maintained, until no visible cellular material and cellular debrisremain.

In certain embodiments, the amniotic membrane is dried immediately(i.e., within 30 minutes) after the decellularization and washing steps.Alternatively, when further processing is not done immediately, theamniotic membrane may be refrigerated, e.g., stored at a temperature ofabout 1° C. to about 20° C., preferably from about 2° C. to about 8° C.,for up to 28 days prior to drying. When the decellularized amnioticmembrane is stored for more than three days but less than 28 days, thesterile solution covering the amniotic membrane is preferably changedperiodically, e.g., every 1-3 days.

In certain embodiments, when the amniotic membrane is not refrigeratedafter washing, the amniotic membrane is washed at least 3 times prior toproceeding to Step IV of the preparation. In other embodiments, when theamniotic membrane has been refrigerated and the sterile solution hasbeen changed once, the amniotic membrane is washed at least twice priorto proceeding to Step IV of the preparation. In yet other embodiments,when the amniotic membrane has been refrigerated and the sterilesolution has been changed twice or more, the amniotic membrane is washedat least once prior to proceeding to Step IV of the preparation.

Prior to proceeding to Step IV, it is preferred that all bacteriologicaland serological testing be assessed to ensure that all tests arenegative.

Step IV.

The final step in this embodiment of the method of collagen biofabricproduction comprises drying the decellularized amniotic membrane of theinvention to produce the collagen biofabric. Any method of drying theamniotic membrane so as to produce a flat, dry sheet of collagen may beused. Preferably, however, the amniotic membrane is dried under vacuum.

In a specific embodiment, an exemplary method for drying thedecellularized amniotic membrane of the invention comprises thefollowing steps:

Assembly of the Decellularized Amniotic Membrane for Drying.

The decellularized amniotic membrane is removed from the sterilesolution, and the excess fluid is gently squeezed out. Thedecellularized amniotic membrane is then gently stretched until it isflat with the fetal side faced in a downward position, e.g., on a tray.The decellularized amniotic membrane is then flipped over so that fetalside is facing upwards, and placed on a drying frame, preferably aplastic mesh drying frame (e.g., Quick Count® Plastic Canvas, Uniek,Inc., Waunakee, Wis.). In other embodiments, the drying frame may be anyautoclavable material, including but not limited to a stainless steelmesh. In a most preferred embodiment, about 0.5 centimeter of theamniotic membrane overlaps the edges of the drying frame. In certainembodiments, the overlapping amniotic membrane extending beyond thedrying frame is wrapped over the top of the frame, e.g., using a clampor a hemostat. Once the amniotic membrane is positioned on the dryingframe, a sterile gauze is placed on the drying platform of a heat dryer(or gel-dryer) (e.g., Model 583, Bio-Rad Laboratories, 200 Alfred NobelDrive, Hercules, Calif. 94547), so that an area slightly larger than theamniotic membrane resting on the plastic mesh drying frame is covered.Preferably, the total thickness of the gauze layer does not exceed thethickness of one folded 4×4 gauze. Any heat drying apparatus may be usedthat is suitable for drying sheet like material. The drying frame isplaced on top of the gauze on the drying platform so that the edges ofthe plastic frame extend above beyond the gauze edges, preferablybetween 0.1-1.0 cm, more preferably 0.5-1.0 cm. In a most preferredembodiment, the drying frame having the amniotic membrane is placed ontop of the sterile gauze with the fetal side of the amniotic membranefacing upward. In some embodiments, another plastic framing mesh isplaced on top of the amniotic membrane. A view of the mesh frame and themembrane dried therein is shown in FIG. 4. In another embodiments, asheet of thin plastic (e.g., SW 182, clear PVC, AEP Industries Inc.,South Hackensack, N.J. 07606) or a biocompatible silicone is placed ontop of the membrane covered mesh so that the sheet extends well beyondall of the edges. In this embodiment, the second mesh frame is notneeded.

In an alternative embodiment, the amniotic membrane is placed one ormore sterile sheets of Tyvek® material (e.g., a sheet of Tyvek® formedical packaging, Dupont Tyvek®, P.O. Box 80705, Wilmington, Del.19880-0705), optionally, with one sheet of Tyvek® on top of the membrane(prior to placing the plastic film). This alternate process will producea smoother version of the biofabric (i.e., without the pattern ofdifferential fiber compression regions along and perpendicular to theaxis of the material), which may be advantageous for certainapplications, such as for example for use as a matrix for expansion ofcells.

Drying the Amniotic Membrane.

In a preferred embodiment, the invention encompasses heat drying theamniotic membrane of the invention under vacuum. While the drying undervacuum may be accomplished at any temperature from about 0° C. to about60° C., the amniotic membrane is preferably dried at between about 35°C. and about 50° C., and most preferably at about 50° C. It should benoted that some degradation of the collagen is to be expected attemperatures above 50° C. The drying temperature is preferably set andverified using a calibrated digital thermometer using an extended probe.Preferably, the vacuum pressure is set to about −22 inches of Hg. Thedrying step is continued until the collagen matrix of the amnioticmembrane is substantially dry, that is, contains less than 20% water byweight, and preferably, about 3-12% water by weight as determined forexample by a moisture analyzer. To accomplish this, the amnioticmembrane may be heat-vacuum dried, e.g., for approximately 60 minutes toachieve a dehydrated amniotic membrane. In some embodiments, theamniotic membrane is dried for about 30 minutes to 2 hours, preferablyabout 60 minutes. Although not intending to be bound by any mechanism ofaction, it is believed that the low heat setting coupled with vacuumpressure allows the amniotic membrane to achieve the dehydrated statewithout denaturing the collagen.

After completion of the drying process in accordance with the invention,the amniotic membrane is cooled down for approximately two minutes withthe vacuum pump running

Packaging and Storing of the Amniotic Membrane.

Once the amniotic membrane is dried, the membrane is gently lifted offthe drying frame. “Lifting off” the membrane may comprise the followingsteps: while the pump is still running, the plastic film is gentlyremoved from the amniotic membrane starting at the corner, while holdingthe amniotic membrane down; the frame with the amniotic membrane islifted off the drying platform and placed on a cutting board with theamniotic membrane side facing upward; an incision is made, cutting alongthe edge 1-2 mm away from the edge of the frame; the amniotic membraneis then peeled off the frame. Preferably, handling of the amnioticmembrane at this stage is done with sterile gloves.

The amniotic membrane is placed in a sterile container, e.g., peelpouch, and is sealed. The biofabric produced in accordance with themethods of the invention may be stored at room temperature for anextended period of time as described supra.

In alternative embodiments, the invention provides a method of preparinga collagen biofabric comprising a chorionic membrane, or both achorionic membrane and an amniotic membrane. It is expected that themethods described above would be applicable to the method of preparing abiofabric comprising a chorionic membrane, or both a chorionic membraneand an amniotic membrane. In one embodiment, the invention encompassesthe use of a collagen biofabric prepared by providing a placentacomprising an amniotic membrane and a chorionic membrane; separating theamniotic membrane from the chorionic membrane; and decellularizing thechorionic membrane. In a specific embodiment, the method further entailswashing and drying the decellularized chorionic membrane. In anotherembodiment, the invention encompasses the use of a collagen biofabricprepared by providing a placenta comprising an amniotic membrane and achorionic membrane, and decellularizing the amniotic and chorionicmembranes. In a specific embodiment, the method further entails washingand drying the decellularized amniotic and chorionic membranes.

4.2.5 Storage and Handling of Collagen Biofabric

Dehydrated collagen biofabric may be stored, e.g., as dehydrated sheets,at room temperature (e.g., 25° C.) prior to use. In certain embodiments,the collagen biofabric can be stored at a temperature of at least 10°C., at least 15° C., at least 20° C., at least 25° C., or at least 29°C. Preferably, collagen biofabric, in dehydrated form, is notrefrigerated. In some embodiments, the collagen biofabric may berefrigerated at a temperature of about 2° C. to about 8° C. Thebiofabric produced according to the methods of the invention can bestored at any of the specified temperatures for 12 months or more withno alteration in biochemical or structural integrity (e.g., nodegradation), without any alteration of the biochemical or biophysicalproperties of the collagen biofabric. The biofabric can be stored forseveral years with no alteration in biochemical or structural integrity(e.g., no degradation), without any alteration of the biochemical orbiophysical properties of the collagen biofabric. The biofabric may bestored in any container suitable for long-term storage. Preferably, thecollagen biofabric of the invention is stored in a sterile doublepeel-pouch package.

The collagen biofabric may be hydrated prior to use. The collagenbiofabric can be rehydrated using, e.g., a sterile physiological buffer.In a specific embodiment, the sterile saline solution is a 0.9% NaClsolution. In some embodiments the sterile saline solution is buffered.In certain embodiments, the hydration of the collagen biofabric of theinvention requires at least 2 minutes, at least 5 minutes, at least 10minutes, at least 15 minutes, or at least 20 minutes. In a preferredembodiment, the hydration of the collagen biofabric of the invention iscomplete within 5 minutes. In yet another preferred embodiment, thehydration of the collagen biofabric of the invention is complete within10 minutes. In yet another embodiment, the hydration of the collagenbiofabric of the invention takes no more than 10 minutes. Once hydrated,the collagen biofabric may be maintained in solution, e.g., sterile 0.9%NaCl solution, for up to six months, with a change of solution, e.g.,every three days.

4.2.6 Sterilization

Sterilization of the biofabric may be accomplished by anymedically-appropriate means, preferably means that do not significantlyalter the tertiary and quaternary structure of the amniotic membraneproteins. Sterilization may be accomplished, for example, using gas,e.g., ethylene dioxide. Sterilization may be accomplished usingradiation, for example, gamma radiation, and is preferably done byelectron beam irradiation using methods known to one skilled in the art,e.g., Gorham, D. Byrom (ed.), 1991, Biomaterials, Stockton Press, NewYork, 55-122. Any dose of radiation sufficient to kill at least 99.9% ofbacteria or other potentially contaminating organisms is within thescope of the invention. In a preferred embodiment, a dose of at least18-25 kGy is used to achieve the terminal sterilization of thebiofabric.

4.2.7 Laminates

The collagen biofabric may be laminated to provide greater stiffness anddurability during the healing process (typically about three months).The collagen biofabric may be laminated as follows.

Collagen biofabric is typically laminated by stacking 2 or more layersof collagen biofabric one atop the other and sealing or drying. Thecollagen biofabric may be laminated either dry or after rehydration.Alternatively, two or more layers of, e.g., amniotic membrane may belaminated prior to initial drying after cell removal, e.g., via a cellscraping step (see Examples, below). If laminated prior to the initialdrying, 2 or more collagen biofabric layers may be stacked one atop theother and subsequently dried, using, for example, a freeze-dryingprocess, or drying under moderate heat with or without vacuum. The heatapplied preferably is not so intense as to cause breakdown ordecomposition of the protein components, especially the collagen, of thecollagen biofabric. Typically, the heat applied is no more than about70° C., preferably no more than about 60° C., and, more preferably, isapproximately 50° C. Lamination time varies with, e.g., the number oflayers being laminated, but typically takes 1-2 hours at 50° C. for thesize pieces of collagen biofabric used for tympanic membrane repair.Preferably, the collagen biofabric laminate comprises 2-6 layers ofcollagen biofabric. In one preferred embodiment, the collagen biofabriclaminate has two layers and is approximately 50 micrometers inthickness. In another embodiment, the collagen biofabric laminate hastwo layers and has a thickness of about 20-60 microns. Preferably, eachof the layers is from the same collagen biofabric lot, that is, the sameplacenta.

The collagen biofabric may also be laminated using an adhesive appliedbetween 2 or more layers of collagen biofabric or amniotic membrane.Such an adhesive is preferably appropriate for medical applications, andcan comprise a natural biological adhesive, for example fibrin glue, asynthetic adhesive, or combinations thereof. The adhesive may further bechemically converted from precursors during the lamination process.

4.2.8 Stem Cells

The collagen biofabric used in the treatment methods described hereinmay also comprise stem or progenitor cells. Preferably, the collagenbiofabric comprises mesenchymal or mesenchymal-like stem cells, forexample, those described in U.S. Pat. Nos. 5,486,359, 6,261,549 and6,387,367, or placenta-derived stem cells such as those described inU.S. Pat. No. 7,045,148 or in U.S. Application Publication Nos.2003/0032179 and 2003/0180269. However, the collagen biofabric maycomprise stem or progenitor cells, preferably mammalian stem orprogenitor cells, from any tissue source. The collagen biofabric maycomprise embryonic stem cells or embryonic germ cells.

The combination of collagen biofabric and stem or progenitor cells maybe accomplished prior to or during application of the collagen biofabricto the leg ulcer. For example, a sheet or piece of collagen biofabricmay be prepared immediately prior to application on the venous leg ulcerby disposing on the surface of the collagen biofabric a solution of stemor progenitor cells and allowing the stem or progenitor cells sufficienttime to attach to the collagen biofabric. The stem or progenitor cells,alternately, may be disposed onto the surface of the collagen biofabric30 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24 ormore hours prior to application of the collagen biofabric onto the legulcer. The number of stem or progenitor cells disposed onto the surfaceof the collagen biofabric may vary, but may be at least 1×10⁶, 3×10⁶,1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹, 1×10¹⁰, 3×10¹⁰, 1×10¹¹,3×10¹¹, or 1×10¹²; or may be no more than 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷,1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹, 1×10¹⁰, 3×10¹⁰, 1×10¹¹, 3×10¹¹, or 1×10¹²stem or progenitor cells. Alternatively, in another embodiment, the stemor progenitor cells, in the number indicated above, may be disposed onthe surface of the collagen biofabric after the collagen biofabric hasbeen applied to the leg ulcer. In another embodiment, the stem cells areapplied directly to the leg ulcer in any of the amounts indicated above,and the leg ulcer is covered with the collagen biofabric. In a morespecific embodiment, the stem cells are applied in aphysiologically-acceptable liquid, such as a saline solution, orembedded in a physiologically-acceptable gel, such as a hydrogel, inwhich the stem or progenitor cells may be maintained and migratethrough. The stem cells, prior to or after contacting with the legulcer, may be contacted with one or more differentiation-modulatingagents, for example, the differentiation-modulating agents described inU.S. Application Publication Nos. 2003/0235909, 2004/0028660, orInternational Application Publication No. WO 03/087333. Methods ofdifferentiating stem cells to, for example, epidermal, mesodermal, andother cell types are known in the art, and are described, e.g., in U.S.Application Publication No. 2004/0028660.

4.3 Kits

Collagen biofabric, useful for the methods of leg ulcer treatment of thepresent invention may be provided in a wrapping or container as part ofa kit for the facilitation of the treatment of leg ulcers. In a specificembodiment, the collagen biofabric is provided an a sterile double-peelpackage. In a more specific embodiment, the collagen biofabric is about6×8 cm. The kit may comprise one or more pieces of collagen biofabricand any other medical device, disposable or drug that would facilitatetreatment of a leg ulcer. Preferably, each piece of the collagenbiofabric in the kit is provided as a single sheet or patch in a sterilecontainer or wrapping separate from the remainder of kit contents. Inanother embodiment, the kit comprises two or more pieces of collagenbiofabric, separately wrapped or contained. In another embodiment, saidkit comprises a support for the collagen biofabric. In specificembodiments, the support may be a natural or a synthetic material. Inother specific embodiments, said support is a plastic film, plasticsheet, or a stretchable plastic wrap. In another embodiment, said kitcomprises one or more disposables. In a specific embodiment, saiddisposables are bandages, means for sterilizing the skin surrounding aleg ulcer, swabs, gloves, or sterile sheets. In another embodiment, saidkit comprises an anti-infective agent. In a specific embodiment, theanti-infective agent is an antibiotic ointment, cream, or spray. Inanother embodiment, said kit comprises a piece of collagen biofabric andone or more wound healing agents. In a specific embodiment, said woundhealing agent is PDGF, TGF, hyaluronic acid, fibrin, or fibronectin. Inanother embodiment, said kit comprises collagen biofabric and a meansfor applying compression to the lower leg. In a specific embodiment, ofany of the kits above, the kit comprises an instruction sheet suitablefor use by a non-medical end user; an instruction sheet suitable for useby an end user in a medical profession; or a materials safety datasheet; or a combination thereof

5. EXAMPLES 5.1 Example 1 Method of Making Collagen Biofabric Materials

The following materials were used in preparation of the collagenbiofabric.

Materials/Equipment

-   -   Copy of Delivery Record    -   Copy of Material/Family Health History/Informed Consent    -   Source Bar Code Label (Donor ID number)    -   Collection # (A sequential number is assigned to incoming        material)    -   Tissue Processing Record (Document ID #ANT-19F); a detailed        record of processing of each lot number is maintained    -   Human Placenta (less than 48 hours old at the start of        processing)    -   Sterile Surgical Clamps/Hemostats    -   Sterile Scissors    -   Sterile Scalpels    -   Sterile Steri-Wrap sheets    -   Sterile Cell Scraper (Nalgene NUNC Int. R0896)    -   Sterile Gauze (non-sterile PSS 4416, sterilized)    -   Sterile Rinsing Stainless Steel Trays    -   Disinfected Processing Stainless Steel Trays    -   Disinfected Plastic Bin    -   Sterile 0.9% NaCl Solution (Baxter 2F7124)    -   Sterile Water (Milli Q plus 09195 or Baxter 2F7113)    -   Sterile Specimen Containers (VWR 15704-014)    -   Personal Protective Equipment (including sterile and non-sterile        gloves)    -   Certified Clean Room    -   Previously Prepared Decellularizing Solution (D-cell); 0.01-1%        deoxycholic acid sodium monohydrate    -   Disinfected Bin    -   Rocking Platform (VWR Model 100)    -   Timer (VWR 21376890)    -   Disinfected Plastic Frame Mesh    -   PVC Wrap Film    -   Vacuum Pump (Schuco-Vac 5711-130)    -   Gel Dryer (i.e., heat dryer; BioRad Model 583)    -   Disinfected Stainless Steel Cutting Board    -   Pouches for Packaging    -   Sterile Stainless Steel Ruler (General Tools MFG. Co 1201)    -   Traceable Digital Thermometer (Model 61161-364, Control Company)    -   Accu-Seal Automatic Sealer (Accu-Seal, Model 630-1B6)

The expectant mother was screened at the time of birth for communicablediseases such as HIV, HBV, HCV, HTLV, syphilis, CMV and other viral andbacterial pathogens that could contaminate the placental tissues beingcollected. Only tissues collected from donors whose mothers testednegative or non-reactive to the above-mentioned pathogens were used toproduce the collagen biofabric.

Following normal birth, the placenta, umbilical cord and umbilical cordblood were spontaneously expelled from the contracting uterus. Theplacenta, umbilical cord, and umbilical cord blood were collectedfollowing birth. The materials were transported to the laboratory wherethey were processed under aseptic conditions in a Clean room having aHEPA filtration system, which was turned on at least one hour prior toprocessing. Gloves (sterile or non-sterile, as appropriate) were worn atall times while handling the product. All unused (waste) segments of theamnion/chorion and contaminated liquids generated during tissueprocessing were disposed of as soon as feasible.

Step I.

A sterile field was set up with sterile Steri-Wrap sheets and thefollowing instruments and accessories for processing were placed on it.

-   -   sterile tray pack    -   sterile Cell Scraper    -   sterile scalpel    -   disinfected processing tray

Sterile pack ID # was recorded in the Processing Record.

The placenta was removed from the transport container and placed ontothe disinfected stainless steel tray. Using surgical clamps andscissors, the umbilical cord was cut off approximately 2 inches from theplacental disc. The umbilical cord was placed into a separate sterilecontainer for further processing. The container was labeled with TissueID Bar Code; and the material and storage solution(s) present (e.g.,type of media) were identified. In some cases, the umbilical cord wasdiscarded if not requested for other projects.

Starting from the edge of the placental membrane, the amnion wasseparated from the chorion using blunt dissection with fingers. This wasdone prior to cutting the membrane.

After the amnion was separated from the entire surface of the chorionand placental disc, the amniotic membrane was cut around the umbilicalcord stump with scissors and detached from the placental disc. In someinstances, if the separation of the amnion and chorion was not possiblewithout tearing the tissue, the amnion and chorion were cut from theplacental disc as one piece and then peeled apart.

The chorion was placed into a separate specimen container to be utilizedfor other projects. The container was labeled with the Tissue ID BarCode, the material and storage solution(s) present (e.g., type of media)were identified, initialed and dated.

If any piece of amnion was still attached to the placental disc it waspeeled from the disc and cutting off around the umbilical cord withscissors. The placenta was placed back into the transport container tobe utilized for other projects.

The appropriate data was recorded in the Tissue Processing Record.

The amniotic membrane was kept in the tray with sterile 0.9% NaClsolution. Preferably, the amniotic membrane is stored by refrigerationfor a maximum of 72 hours from the time of delivery prior to the nextstep in the process.

Step II.

The amniotic membrane was removed from the specimen container one pieceat a time and placed onto the disinfected stainless steel tray. Otherpieces were placed into a separate sterile stainless steel tray filledwith sterile water until they were ready to be cleaned. Extra pieces ofamnion from the processing tray were removed and placed in a separaterinsing stainless steel tray filled with sterile water.

The amniotic membrane was rinsed with sterile water if grosslycontaminated with blood maternal or fetal fluids/materials changingsterile water as needed.

The amniotic membrane was placed on the processing tray with thematernal side facing upward. Using a sterile Cell Scraper, as much aspossible of visible contamination and cellular material from thematernal side of the amnion was carefully removed. (Note: minimalpressure should be applied for this step to prevent tearing themembrane). Sterile water was used to aid in the removal of cells andcellular debris. The amniotic membrane was further rinsed with sterilewater in the separate sterile stainless steel rinsing tray.

The amniotic membrane was turned over so that the fetal side was facingupward and placed back on the processing tray and rinsed with sterilewater. Visible cellular material and debris using the Cell Scraper wasgently removed (Note: minimal pressure should be applied for this stepto prevent tearing the membrane). Sterile water was used to aid in theremoval of cells and cellular debris.

The amniotic membrane was rinsed with sterile water in between cleaningrounds in separate sterile rinsing trays. The tissue was cleaned as manytimes (cleaning rounds) as necessary to remove most if not all ofvisible cellular material and debris from both sides of the membrane.The sterile water was changed in the rinsing trays in between rinses.

The processing tray was rinsed with sterile water after each cleaninground.

All other pieces of amnion were processed in the same manner and placedinto the same container. Tissue Id Bar Code was affixed, the materialand storage solution(s) present (e.g., type of media) were identified,initials date were added.

The appropriate information and the date were recorded in the TissueProcessing Record.

Step III.

The amniotic membrane was removed from the rinsing tray, (or fromstorage container) excess fluid was gently squeezed out with fingers andthe membrane was placed into the sterile specimen container. Thecontainer was filled up to the 150 ml mark with D-cell solution ensuringthat all of the amniotic membrane was covered and the container wasclosed.

The container was placed in the bin on the rocking platform. The rockingplatform was turned on and the membrane was agitated in D-cell solutionfor a minimum of 15 minutes and a maximum of 120 minutes at Setting #6.

A new sterile field was set up with new sterile instruments anddisinfected tray in a same manner as in the Step I. Sterile pack ID #was recorded in the Processing Record.

After agitation was completed, the rocking platform was turned off andthe membrane was removed from the container. The membrane was placedinto a new sterile stainless steel processing tray. Sterile 0.9% NaClsolution was added to cover the bottom of the tray.

Using a new sterile Cell Scraper, residual D-cell and cellular material(if any) was removed from both sides of the tissue. This step wasrepeated as many times as needed to remove as much as possible ofvisible residual cellular material from the entire surface on bothsides. The membrane was rinsed with sterile 0.9% NaCl solution in aseparate rinsing tray in between cleaning rounds. The sterile 0.9% NaClsolution was changed in the rinsing trays in between rinses.

After the last cleaning round was completed, the membrane was rinsedwith sterile 0.9% NaCl solution and placed into the new sterile specimencontainer filled with sterile 0.9% NaCl solution.

All remaining pieces of amniotic membrane were processed in exactly thesame manner.

When all amniotic membrane pieces were processed and in the containerwith the sterile 0.9% NaCl solution, the container was placed in the binon the rocking platform to agitate for a minimum of 5 minutes at setting#6. After agitation was completed, the membrane was removed from thespecimen container, the sterile 0.9% NaCl solution was changed in thecontainer and the membrane was placed back into the specimen container.

The specimen container was labeled with Tissue ID Bar Code andQuarantine label. The material and storage solution(s) present (e.g.,type of media) were identified, initialed and dated. The specimencontainer was placed into a clean zip-lock bag and placed in therefrigerator (2-8° C.).

All appropriate data was recorded in the Tissue Processing Record.

When serology results became available, the appropriate label (SerologyNegative or For Research Use Only) was placed on the top of theQuarantine label and those containers were segregated from Quarantinedones.

Step IV.

Before proceeding with Step IV, the Tissue Status Review was checked tomake sure all applicable test results were negative.

A sterile field was set up with sterile Steri-Wrap sheet and all sterileand disinfected instruments and accessories were set up in the samemanner as in Steps II and III.

The membrane was removed from the refrigerator and placed into a newsterile stainless steel processing tray. Sterile 0.9% NaCl solution wasadded to cover the bottom of the tray.

All visible cellular material and debris (if any) was gently removedusing a new sterile Cell Scraper (Note: minimal pressure should beapplied for this step to prevent tearing the membrane). Sterile 0.9%NaCl solution was used to aid in removal of the cells and debris.

The membrane was rinsed in the separate sterile stainless steel rinsingtray filled with the sterile 0.9% NaCl Solution. 0.9% NaCl Solution waschanged in between cleaning rounds. The membrane was placed into a newsterile specimen container, the container was filled with fresh sterile0.9% NaCl solution and placed on the rocking platform for agitation fora minimum of 5 minutes at Setting #6.

The previous step was repeated 3 times and the sterile 0.9% NaClsolution was changed in between each agitation. Appropriate data wasrecorded in the Tissue Processing Record.

The membrane was removed from the specimen container one piece at atime, excess fluid was gently squeezed out with fingers and the membranewas placed onto a sterile processing tray. The membrane was gentlystretched until flat; ensuring it was fetal side down.

The frame was prepared by cutting the disinfected plastic sheet withsterile scissors. The size of the frame should be approximately 0.5 cmsmaller in each direction than the membrane segment. The frame wasrinsed in the rinsing tray filled with sterile 0.9% NaCl solution.

The frame was placed on the slightly stretched membrane surface andpressed on it gently. It is imperative that the smooth side of theplastic frame faces the tissue.

Using a scalpel, the membrane was cut around the frame leavingapproximately 0.5 cm extending beyond frame edges. The excess membranewas placed back into the specimen container

The membrane edges that are extended beyond the frame were wrapped overthe edges of the frame using clamps or tweezers and put aside on thesame tray.

The next piece of membrane was processed in the same manner. It ispreferred that the total area to be dried does not exceed 300 cm² perheat dryer. While ‘framing out’ the piece of membrane, it is preferredthat the non-framed pieces remain in the container in sterile 0.9% NaClsolution.

The drying temperatures of dryers were set and verified using acalibrated digital thermometer with extended probe. The dryingtemperature was set at 50° C. The data was recorded in the TissueProcessing Record.

The vacuum pump was turned on.

A sterile gauze was placed on the drying platform of the heat dryer,covering an area slightly larger than the area of the framed membrane.It is important to make sure that the total thickness of the gauze layerdoes not exceed thickness of one folded 4×4 gauze.

One sheet of plastic framing mesh was placed on top of the gauze. Theplastic mesh edges should extend approximately 0.5-1.0 cm beyond gauzeedges.

The framed membrane was gently lifted and placed on the heat dryerplatform on top of the plastic mesh with the membrane side facingupward. This was repeated until the maximum amount of membrane (withoutexceeding 300 cm²) was on the heat dryer platform. (NOTE: fetal side ofthe amnion is facing up).

A piece of PVC wrap film was cut large enough to cover the entire dryingplatform of the heat dryer plus an extra foot.

With the vacuum pump running, the entire drying platform of the heatdryer was gently covered with the plastic film leaving ½ foot extendingbeyond drying platform edges on both sides. Care was taken that the filmpull tightly against the membrane and frame sheet (i.e., it is “suckedin” by the vacuum) and that there were no air leaks and no wrinkles overthe tissue area). The lid was subsequently closed.

The vacuum pump was set to approximately −22 inches Hg of vacuum. Thepump gage was recorded after 2-3 min of drying cycle. The membrane washeat vacuum dried for approximately 60 minutes. Approximately 15-30minutes into the drying process, the sterile gauze layer was replaced inthe heat dryer with a new one. The total thickness of the gauze layermust not exceed thickness of one folded 4×4 gauze.

After the change, care was taken so that the plastic film pulled tightlyagainst the membrane and the frame sheet and there were no air leaks andno wrinkles over the membrane area.

The integrity of the vacuum seal was periodically checked by checkingthe pump pressure monometer. After completion of the drying process, theheat dryer was opened and the membrane was cooled down for approximatelytwo minutes with the pump running

A new sterile field was set up with sterile Steri-wrap and disinfectedstainless steel cutting board underneath it. As this point sterilegloves were used. With the pump still running, the plastic film wasgently removed from the membrane sheet starting at the corner andholding the membrane sheet down with a gloved hand. The frame was gentlylifted with the membrane off the drying platform and placed on thesterile field on the top of the disinfected stainless steel cuttingboard with the membrane side facing upward. Using a scalpel, themembrane sheet was cut through making an incision along the edge 1-2 mmaway from the edge of the frame. The membrane was held in place with agloved (sterile glove) hand. Gently the membrane sheet was lifted off ofthe frame by peeling it off slowly and then placed on the sterile fieldon the cutting board.

Using scalpel or sharp scissors, the membrane sheet was cut intosegments of specified size. All pieces were cut and secured on thesterile field before packaging. A single piece of membrane was placedinside the inner peel-pouch package with one hand (sterile) whileholding the pouch with another hand (non-sterile). Care was taken not totouch pouches with ‘sterile’ hand. After all pieces were inside theinner pouches they were sealed. A label was affixed with the appropriateinformation (e.g., Part #, Lot #, etc.) in the designated area on theoutside of the pouch. All pieces of membrane were processed in the samemanner. The labeled and sealed peel-pouch packages were placed in thewaterproof zip-lock bag for storage until they were ready to be shippedto the sterilization facility or distributor. All appropriate data wererecorded on the Tissue Processing Record.

5.2 Example 2 Alternative Method of Making Collagen Biofabric

A placenta is prepared substantially as described in Step I of Example 1using the Materials in that Example. An expectant mother is screened atthe time of birth for communicable diseases such as HIV, HBV, HCV, HTLV,syphilis, CMV and other viral and bacterial pathogens that couldcontaminate the placental tissues being collected. Only tissuescollected from donors whose mothers tested negative or non-reactive tothe above-mentioned pathogens are used to produce the collagenbiofabric.

A sterile field is set up with sterile Steri-Wrap sheets and thefollowing instruments and accessories for processing were placed on it:sterile tray pack; rinsing tray, stainless steel cup, clamp/hemostats,tweezers, scissors, gauze.

The placenta is removed from the transport container and placed onto adisinfected stainless steel tray. Using surgical clamps and scissors,the umbilical cord is cut off approximately 2 inches from the placentaldisc.

Starting from the edge of the placental membrane, the amnion isseparated from the chorion using blunt dissection with fingers. This isdone prior to cutting the membrane. After the amnion is separated fromthe entire surface of the chorion and placental disc, the amnioticmembrane is cut around the umbilical cord stump with scissors anddetached from the placental disc. In some instances, if the separationof the amnion and chorion is not possible without tearing the tissue,the amnion and chorion is cut from the placental disc as one piece andthen peeled apart.

The appropriate data is recorded in the Tissue Processing Record.

The amniotic membrane is rinsed with sterile 0.9% NaCl solution toremove blood and fetal fluid or materials. The saline solution isreplaced as necessary during this rinse.

The amnion is then placed in a 0.9% saline, 1.0% deoxycholic acidsolution in a specimen container and refrigerated at 2-8° C. for up to15 days, with changes of the solution every 3-5 days. During or at theend of incubation, the serological tests noted above are evaluated. Ifthe tests indicate contamination with one or more pathogens, the amnionis rejected and processed no further. Tissue indicated as derived from aCMV-positive donor, however, is still suitable for production ofbiofabric.

Once the incubation is complete, the amnion is removed from the specimencontainer, placed in a sterile tray and rinsed three times with 0.9%NaCl solution to reduce the deoxycholic acid from the tissue. With theamnion placed maternal side up, the amnion is gently scraped with a cellscraper to remove as much cellular material as possible. Additionalsaline is added as needed to aid in the removal of cells and cellulardebris. This step is repeated for the fetal side of the amnion. Scrapingis followed by rinsing, and is repeated, both sides, as many times asnecessary to remove cells and cellular material. The scraped amnion isrinsed by placing the amnion in 0.9% saline solution a separatecontainer on a rocking platform for 5-120 minutes at setting #6. Thesaline solution is replaced, and the rocking rinse is repeated.

After rinsing is complete, the amnion is optionally stored in a zip-lockbag in a refrigerator.

The scraped amnion is then placed fetal side down onto a sterileprocessing tray. The amnion is gently massaged by hand to remove excessliquid, and to flatten the membrane. A sterile plastic sheet is cut sothat its dimensions are approximately 0.5 cm smaller in each directionthan the flat amnion. This plastic sheet is briefly rinsed in 0.9% NaClsolution. The plastic sheet is placed, smooth side down, on theflattened amnion, leaving a margin of uncovered amnion. A scalpel isused to trim the amnion, leaving approximately 0.5 cm extending beyondthe sheet edges. These extending amnion edges are wrapped back over theplastic sheet. The total tissue area to be dried does not exceed 300 cm²for a standard vacuum heat dryer.

A sheet of sterile gauze is placed in a vacuum heat dryer. A thinplastic mesh is placed on the gauze so that approximately 0.5-10.0 cmextends beyond the edges of the gauze. The amnion and plastic sheet arethen placed into the vacuum heat dryer on top of the mesh, tissue sideup, and the amnion is covered with a sheet of PVC wrap film. The dryeris set at 50° C., and the temperature is checked periodically to ensuremaintenance of 50° C.±1° C. The vacuum pump is then turned on and set toapproximately −22 inches Hg vacuum. Drying is allowed to proceed for 60minutes.

The dried amnion is then stored in a sealed plastic container forfurther use.

5.3 Example 3 Collagen Biofabric Laminate

The collagen biofabric produced by the methods described in Example 1,above, was laminated as follows. “Dry” collagen biofabric was producedby the procedure outlined in Example 1, above, then rehydrated andlaminated. “Wet” collagen biofabric was prepared up to Step III ofExample 1 (that is up to the point of vacuum drying), then laminated.After mounting frames were cut and the “dry” biofabric was rehydrated,both types of biofabric were mounted by placing the fetal side down,placing the mounting frame on top of the tissue, and cutting the tissue,leaving about 1 cm edge around the frame. The 1 cm edge was folded overthe edge of the frame using a cell scraper. These steps were repeatedfor adding additional pieces of collagen biofabric. The laminatedbiofabric was then placed in a gel dryer and dried to substantialdryness (<20% water content by weight). Laminates were then cut to 2×6cm samples.

Separate lots of the laminated collagen biofabric were evaluated asfollows. Dimensions of dry (DT) and wet (WT) laminated collagenbiofabric were determined for laminates containing 2, 3, 5 or 8 layers,as shown in Table 1:

TABLE 1 Thickness (μm) Length (mm) Width (mm) Weight (mg) DT2  29 ± 1220.0 ± 0.3 5.2 ± 0.1 0.87 ± 0.02 DT3 32 ± 2 20.5 ± 0.1 5.2 ± 0.2 1.26 ±0.11 WT2  20 ± 15 20.2 ± 0.2 5.0 ± 0.3 0.93 ± 0.17 WT3 15 ± 5 19.6 ± 0.15.1 ± 0.3  0.9 ± 0.04 WT5 31 ± 5 19.8 ± 0.4 5.3 ± 0.1 2.06 ± 0.2  WT8115 ± 26 20.3 ± 0.2 5.1 ± 0.4 4.92 ± 0.56Specimens showed no signs of delamination over the first two dayspost-lamination, when kept under dry conditions at room temperature. Thelaminated collagen biofabric additionally showed no signs ofdelamination when kept in stirred 0.9% saline, room temperature, for tendays.

Larger laminated collagen biofabric specimens were tested for laminatedurability and resistance to delamination. 1×2 cm specimens from thelist listed above (i.e., DT2, DT3, WT2, WT3, WT5 and WT8) were placed inPetri dishes in 5 ml phosphate buffered saline. The specimens were lefton an orbital shaker for approximately 24 hours at 95 RPM. Nodelamination of the specimens was observed, either during shaking orthereafter during simple handling.

5.4 Example 4 Methods of Treatment

A patient presents with a venous leg ulcer on the inside ankle area ofthe right leg. The leg ulcer is evaluated for infection. The leg ulceris debrided after administration of a local anesthetic. The leg ulcer ismeasured, and a small sheet of collagen biofabric is selected that willcompletely cover the leg ulcer. The collagen biofabric is placed on theleg ulcer and gently pressed into the leg ulcer so that the collagenbiofabric contacts substantially all of the ulcerated tissue with no oronly small air pockets. The collagen biofabric is allowed to adhere tothe leg ulcer by adsorption to the ulcer. The collagen biofabric is thencovered with a dressing to prevent disturbance of the leg ulcer. Asecond sheet of collagen biofabric is placed on the healing wound 4-5weeks after the first application, in the same manner as the firstapplication. If necessary, a third application is performed at the 8-9week mark, and every 3-4 thereafter as needed. The leg ulcer isevaluated every 3-4 weeks during the wound healing process.

A second patient presents with a venous leg ulcer on the inside anklearea of the right leg. The leg ulcer is evaluated for infection. The legulcer is debrided after administration of a local anesthetic. The legulcer is measured, and a small sheet of collagen biofabric is selectedthat will completely cover the leg ulcer. The collagen biofabric isplaced on the leg ulcer and gently pressed into the leg ulcer so thatthe collagen biofabric contacts substantially all of the ulceratedtissue with no or only small air pockets. The collagen biofabric isallowed to adhere to the leg ulcer by adsorption to the ulcer. Thecollagen biofabric, and the lower leg, is then covered with acompression bandage. The compression bandage is maintained on the legfor the duration of the healing process, with changes as necessary. Asecond sheet of collagen biofabric is placed on the healing wound 4-5weeks after the first application, in the same manner as the firstapplication. If necessary, a third application is performed at the 8-9week mark, and every 3-4 thereafter as needed.

5.5 Example 5 Treatment of Venous Leg Ulcer Patients with CollagenBiofabric

Several individuals having leg ulcers were treated with collagenbiofabric in a non-clinical setting as follows.

Patient 1: Patient 1 presented with two venous leg ulcers on the ankle,one approximately 1 cm×1.5 cm, the other approximately 1.8 cm×1.3 cm. A6×8 dry sheet of collagen biofabric was laid over both, and allowed toadhere by adsorption to the wound exudates. By six weekspost-application, the ulcers were completely healed.

Patient 2: Patient 2 presented with venous leg ulcers measuringapproximately 2.2 cm×1.7 cm and 0.4 cm×1.0 cm. A 6×8 dry sheet ofcollagen biofabric was laid over both, and allowed to adhere byadsorption to the wound exudates. Two weeks after the initial sheet wasapplied, a second sheet was applied to the ulcers. A third applicationof a collagen biofabric sheet was made five weeks after the initialapplication. The ulcers were substantially healed by nine weeks afterinitial application.

Patient 3: Patient 3 presented with a venous leg ulcer, locatedproximate to the ankle, measuring 1.5 cm×0.9 cm. Treatment consisted oftwo applications of a dry collagen biofabric sheet, the second fiveweeks after the first. By eight weeks after the first application, theulcer had epithelialized.

Patient 4: Patient 4 presented with a single venous leg ulcer measuringapproximately 3.0 cm×1.5 cm. By six weeks after application of a singlesheet of dry collagen biofabric, the ulcer had shrunk to approximately1.8 cm×0.8 cm.

Patient 5: Patient 5 presented with a single venous leg ulcer measuringapproximately 2.0 cm×1.2 cm. A single dry sheet of collagen biofabricwas applied and allowed to allowed to adhere by adsorption to the ulcerexudates. After application, the collagen biofabric was moistened tothat it adhered, as well, to the skin surrounding the ulcer. A dressingand compression were then applied. By 2 weeks post-application, theulcer had noticeably diminished in size.

5.6 Example 6 Observational Use Study of Biofabric

A multicenter observational biofabric use study was conducted. The studyconcluded that collagen biofabric, applied as a dehydrated humanamniotic membrane, is safe when used in the management of non-infected,full- or partial-thickness acute or chronic wounds.

Investigators were asked to enroll any patient who presented with anon-infected, full- or partial thickness wound which could havebenefited from the biofabric, including acute or chronic wounds. As thestudy was observational, patients were not randomized, and there were nocontrol groups. Patients with infected wounds, or with knownhypersensitivity to the biofabric, were excluded from the study.Patients were evaluated for type of wound, safety (including number andtype of adverse events), and efficacy of biofabric treatment. A total of225 patients, exhibiting a total of 240 wounds, were treated in thestudy. A summary of enrolled patients is presented in Table 2. Althoughthe study included patients having burns, collagen vascular diseasewounds and acute wounds, only data relating to leg ulcers is presentedin the tables herein.

TABLE 2 Patient wound types. Age at Age at Number Number First First ofof Males Females Visit Visit Wound Type patients Wounds N (%) N (%)(average) (range) Diabetic foot ulcers 42 45 27 (64%) 15 (36%) 60.619.0-87.5 Pressure ulcers 22 22 15 (68%)  7 (32%) 66.2 30.8-89.8Arterial (ischemic) 11 14  7 (64%)  4 (36%) 71.9 56.4-84.7 ulcers Venousstasis ulcers 78 85 42 (54%) 36 (46%) 64.2 28.7-88.6 N = numberThirty five of the 42 patients with diabetic foot ulcers completed thestudy, as did 17 of the 22 patients with pressure (decubitus) ulcers, 10of the 11 patients with arterial ulcers, and 67 of the 78 patients withvenous leg ulcers.

Each of the patients participating in the study received at least onepiece (6×8 cm) of the biofabric, applied directly to the wound. Thebiofabric was replaced when it appeared to have been absorbed into thewound. Where the wound presented dry, the wound was wetted withphysiological sterile normal saline prior to applying the biofabric. Themaximum number of pieces of biofabric applied to any one patient was 15.The maximum number of weeks any patient was treated with the biofabricwas 27.7. The patient with the highest number of pieces applied for thelongest time was one who had seven pieces applied over 15 weeks oftreatment. Patient exposure to the biofabric is summarized in Table 3:

TABLE 3 Summary of exposure to collagen biofabric. Number Number N NNumber Number of 6 × 8 of 6 × 8 exposed exposed of weeks of weeks piecespieces to 1 to >1 Wound N observed observed applied applied piece pieceType (wounds) (range) (average) (average) (range) biofabric biofabricDiabetic 45 <1-25.3  9.5 2.8  1-15 14 31 foot ulcers Pressure 220.3-27    8.6 2.1 1-7 11 11 ulcers Arterial 14 1-25.5 11.7 2.5 1-6 5 9(ischemic) ulcers Venous 85 1-27.7 8.4 2.4 1-7 32 53 stasis ulcers N =number

During the course of the study, 23 of the leg ulcer patients had adverseevents, including 16 that developed wound infection, three thatdeveloped a worsening wound, three that developed cellulites at thewound site, and one that developed or experienced rash at the woundsite. Six patients with leg ulcers experienced severe adverse events,apparently unrelated to the biofabric, during the course of the study.Three of these patients died, and one was hospitalized, for reasonsunknown to the study organizers; one was hospitalized for peripheralvascular disease; and one experienced a cerebral vascular accident. Inall, none of the adverse or severe adverse events was determined to bedue to the use of the biofabric.

Although this observational study was primarily designed to capturesafety and use information, some efficacy analyses were performed.Specifically, baseline wound surface areas (healers vs. non-healers),decrease in wound surface areas (healers vs. non-healers), and decreasein wound surface area over time were analyzed. Efficacy was determinedby visual inspection of the wound(s) during the study. Patients that hada detectable reduction in the size of their wounds were deemed“healers,” while those whose wounds did not appear to reduce in sizewere deemed “nonhealers”. Table 4 summarizes wound size reduction inhealers vs. nonhealers.

TABLE 4 Baseline wound surface areas by wound type. HEALED NON-HEALEDALL WOUNDS WOUNDS WOUNDS Baseline wound Baseline wound Baseline woundWounds surface area surface area surface area N healed (cm²) (cm²) (cm²)Wound Type (wounds) N (%) Average (range) Average (range) Average(range) Diabetic foot ulcers 45 15 (33%)  8.0 (0.2-81.6)  8.2 (0.2-81.6) 8.2 (0.3-22.5) [All diabetic foot  [6.3 (0.2-22.5)]*   [2.9(0.2-10.3)]* ulcers except one outlier]* Pressure ulcers 22  9 (41%) 4.3(0.75-22) 1.4 (0.75-3) 6.4 (0.8-22) Arterial (ischemic) ulcers 14  5(36%) 20.8 (1.2-108)   3.1 (1.2-6.4) 30.6 (1.4-108) Venous stasis 85 36(42%) 13.3 (0.06-203) 5.2 (0.4-40)  18.8 (0.06-203) ulcers N = number*Upon further clinical evaluation, one wound, classified by theinvestigator as a diabetic foot ulcer with a surface area of 81.6 cm²,appeared to be a post-operative wound located on the calf of a diabeticpatient. Therefore, a separate efficacy calculation was made excludingthis wound.

Decreases in wound surface area for healers, decreases in wound width,and decreases in wound surface area over time observed for all wounds bywound types, are presented in Tables 5-7, below.

TABLE 5 Decrease in wound surface area by wound type (healers) Baselinewound Wounds surface area Weeks to heal Rate of healing N healed (cm²)Average (cm²/week) Wound Type (wounds) N (%) Average (range) (range)Average (range) Diabetic foot 45 15 (33%)  8.2 (0.2-81.6) 8.4 (3-25.3)1.1 (0.03-11.7) ulcers Pressure ulcers 22  9 (41%) 1.4 (0.25-3) 8.3(2-19)  0.23 (0.02-0.43)  Arterial 14  5 (36%)  3.1 (1.2-6.4) 6.0(2.5-12) 0.73 (0.13-1.8)  (ischemic) ulcers Venous stasis 85 36 (42%)5.2 (0.4-40) 7.3 (2-27.7) 1.1 (0.05-10.8) ulcers N = number

TABLE 6 Decrease in wound width (for healers) by wound type BaselineRate of wound width decrease in Wounds (cm) Weeks to heal width N healedAverage Average (cm/week) Wound Type (wounds) N (%) (range) (range)Average (range) Diabetic foot 45 15 (33%)  1.3 (0.4-3.4) 8.4 (3-25.3)0.20 (0.03-0.63) ulcers Pressure ulcers 22  9 (41%) 0.97 (0.5-1.5) 8.3(2-19)  0.17 (0.04-0.33) Arterial 14  5 (36%) 1.36 (0.8-2)  6.0 (2.5-12)0.34 (0.09-0.76) (ischemic) ulcers Venous stasis 85 36 (42%) 1.6(0.4-5)  7.3 (2-277)  0.33 (0.04-1.7)  ulcers N = number

TABLE 7 Decrease in wound surface area over time for all wounds by woundtype. Final wound Baseline wound surface area Weeks Rate of healingsurface area (cm²) observed (cm²/week) N (cm²) Average Average AverageWound Type (wounds) Average (range) (range) (range) (range) Diabeticfoot 45 8.0 (0.20-81.6) 3.4 (0-22.5) 9.5 (0-25.3)  0.6 (−1.8-11.7)ulcers Pressure ulcers 22 4.3 (0.25-22)  2.8 (0-22.5) 8.6 (0.3-27) 0.31(−0.28-2.9) Arterial 14 20.8 (1.2-49)    15.2 (0-82.7)  11.7 (1-25.5) 0.56 (−1.3-1.8)  (ischemic) ulcers Venous stasis 85 13.3 (0.06-202.5)6.8 (0-69.9) 8.4 (1-27.7) 0.80 (−3.2-10.8) ulcers N = number

One wound type subgroup, venous stasis ulcers, was examined in detail.Outcomes for this group are summarized in Table 8.

TABLE 8 Venous stasis ulcer (VSU) summary. Healed VSU Non-healed All VSU(42%) VSU (56%) (N = 85) (N = 36*) (N = 48*) Patients treated (N) 78 N/AN/A Completed patients (N) 67 (85%) 31 36 Discontinued patients^(¶) (N)11 (14%) 0 10 Age (mean years) 64.2 65.3 63.9 Weeks to healing (mean)N/A 7.3 N/A Weeks to healing (median) N/A 5.5 N/A Rate of healing(cm²/week) N/A 1.8 N/A Weeks of observation (mean) 8.4 7.3 9.3 Weeks ofobservation (range) 1-27.7 2-27.7 1-21.6 Mean wound baseline surface13.3 5.2 18.8 area (cm²) (all wounds) Median wound baseline sur- 4.8 2.79.0 face area (cm²) Pieces biofabric used (N) 2.4 2.2 2.5 Wounds treatedwith 1 piece 32 15 16 biofabric (N) # Wounds treated with >1 53 21 32piece biofabric (N) SAEs (N)^(†) 4 0 4 AEs (N)^(‡) 9 1 8 AE = Adverseevent; SEA = severe adverse event; N = number; N/A = not applicable.*One patient was lost to follow-up prior to final wound healingdetermination. ^(¶)Lost to follow up (N = 5), adverse event (N = 5),withdrew consent (N = 1). ^(†)Hospitalization for unknown reason (N =2), death, cause unknown (N = 1). ^(‡)Wound infection (N = 5), worseningwound (N = 1), cellulitis (N = 1), rash (N = 1).

CONCLUSIONS

The collagen biofabric (dried amniotic membrane) was shown to be safelyapplicable to patients having the four major leg ulcer types. Multiplepieces of the biofabric could safely be applied to non-infected,partial- or full-thickness chronic wounds. Leg ulcers, particularlyvenous leg ulcers, can be considered non-healing wounds. Thus, thoughnot an efficacy study, the study data indicate that this collagenbiofabric be useful in the treatment or management of leg ulcers.

EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1.-24. (canceled)
 25. A sterilized, decellularized, and dehydratedamnion-derived biofabric, wherein said biofabric is coated with orimpregnated with granulocyte macrophage stimulating factor (GM-CSF). 26.The biofabric of claim 25, wherein said biofabric comprises nativevascular-endothelial growth factor (VEGF) and fibroblast growth factor(FGF).
 27. A pharmaceutical composition comprising the biofabric ofclaim
 25. 28. A pharmaceutical composition comprising the biofabric ofclaim
 26. 29. The biofabric of claim 25, wherein said biofabric isshaped as a sheet or membrane.
 30. The biofabric of claim 29, whereinsaid biofabric is shaped as a sheet.
 31. The biofabric of claim 25,wherein said biofabric is shaped as a netting or webbing.
 32. Thebiofabric of claim 25, wherein said biofabric is mounted on a support.33. The biofabric of claim 32, wherein said support is a bandage.
 34. Amethod of treating a leg ulcer, comprising contacting said ulcer withthe collagen biofabric of claim
 25. 35. The method of claim 34, whereinsaid contacting improves at least one aspect of the leg ulcer, orprevents or reduces the worsening of at least one aspect of a leg ulcer.36. The method of claim 34, wherein said contacting is for a timesufficient for at least one aspect of the leg ulcer to measurablyimprove compared to a leg ulcer not contacted with the collagenbiofabric.
 37. The method of claim 34, wherein said contacting is for atime sufficient to prevent or reduce the worsening of at least oneaspect of said leg ulcer.
 38. The method of claim 34, wherein said legulcer is a venous leg ulcer, arterial leg ulcer, diabetic leg ulcer, ordecubitus ulcer.
 39. The method of claim 38, wherein said leg ulcer is avenous leg ulcer.
 40. The method of claim 34, wherein said contactingcomprises placing the collagen biofabric on the leg ulcer so thatsubstantially all of the surface area of the biofabric contacts the legulcer.
 41. A kit for the facilitation of the treatment of leg ulcers,comprising the collagen biofabric of claim
 25. 42. The kit of claim 41,further comprising a medical device, disposable or drug that facilitatestreatment of a leg ulcer.
 43. The kit of claim 41, wherein the collagenbiofabric is provided as a single sheet or patch in a sterile containeror wrapping.
 44. The kit of claim 41, further comprising ananti-infective agent and/or a wound healing agent.